Testosterone in the molar vesicle fluid and theca-lutein cyst fluid. Testosterone was estimated by radioimmunoassay in molar vesicle fluid in 41 patients and theca-lutein cyst fluid in three patients with hydatidiform moles. The testosterone concentration in the molar vesicle fluid ranged from 0.4 ng/ml to 28.1 ng/ml with a mean+/-SEM of 6.0+/-1.0 ng/ml while in the theca-lutein cyst fluid, it ranged from 4.7 ng/ml to 23.5 ng/ml. Oestradiol-17beta and human chorionic gonadotrophin (HCG) were also estimated in the molar vesicle fluid. The oestradiol-17beta concentration ranged from 0.1 ng/ml to 26.2 ng/ml with a mean+/-SEM of 10.7+/-1.1 ng/ml. HCG concentration ranged from 18 000 to 150 000 mIU/ml with a mean+/-SEM of 49 800+/-5 000 mIU/ml. A positive correlation was found between testosterone and oestradiol-17beta (r = +0.37; P less than 0.01) but not between testosterone and HCG (r = 0.32; 0.1 greater than P greater than 0.05). Our findings suggest that molar trophoblast is the major source of testosterone.


Hypothyroidism in pregnancy.

Hypothyroidism in pregnancy. Two mothers with hypothyroidism gave birth to normal infants. In one patient there was good evidence that maternal hypothyroidism improved during the last trimester due to placental transfer of fetal thyroid hormone. If pregnancy is advanced when the diagnosis of hypothyroidism is made, it would seem that replacement therapy may be withheld without detriment to the fetus.


Unusual presacral tumours obstructing delivery. Two patients who had obstructed labour due to a presacral tumour are reported. Both tumours were diagnosed in the second stage of labour. In one patient delivery was accomplished with Barton's forceps. The other patient required a Caesarean section. The tumours were excised at  later date. One was an epidermoid cyst and the other was thought to be a malignant haemangioendothelioma.


The tilted disc.

The tilted disc. Sixty tilted discs were studied by colour photography, including some by fluorescein angiography. Attention was drawn to the contracted, D-shaped outline of the scleral canal, and it was suggested that fewer fibres than normal enter the defective side of the disc. This was supported by examination of the nerve fibre layer and the discovery of field defects in 13 of the 27 eyes in which the visual fields were examined. The similarity of these features with congenital hypoplasia of the optic nerve head was noted.


Amblyopia resulting from penalisation: neurophysiological studies of kittens reared with atropinisation of one or both eyes. Atropinisation of the eyes--a clinical method of treating strabismus called "penalisation"--in developing kittens caused a reduction in the spatial resolving power of cells in the lateral geniculate nucleus driven by the penalised eye, regardless of whether 1 eye or both eyes had been atropinised. However, binocularity of cells in the visual cortex was reduced only in monocularly penalised cats. It appears that sharply focused foveal images are important in the development of good visual acuity but synergy of the inputs to the 2 eyes is required for the development of binocular vision.


Contrast threshold of random dot stereograms in anisometropic amblyopia: A clinical investigation. The contrast thresholds of random dot stereograms were obtained for both anisometropic amblyopic and normal patients. The elevation of the contrast threshold found in both amblyopic and non-amblyopic eyes of the anisometropes reflects the relative strength of the binocular inhibition mechanism present. These findings were related to the standard clinical tests used in this investigation and other recent psychophysical and physiological investigations.


Histopathology of human superficial herpes simplex keratitis. In vivo corneal replicas were made in 20 cases of patients with superficial dendritic ulcers of the cornea. Histopathological study of the replicas and superficial epithelial cells showed that the dendrites are composed of rounded epithelial cells and variable sized syncytia containing bizarre shaped nuclei. Pseudopodia-like processes containing DNA and some RNA extend from the syncytia into the surrounding epithelial cells, which on coming into contact with these processes become rounded and liquefied to give rise to another syncytium. The epithelial cells adjacent to the dendrite and elongated and usually orientated parallel to the long axis of the lesion. Surrounding the terminal bulbs, they are disposed in an arcuate fashion. These cells show C-mitotic lesions, intranuclear and cytoplasmic inclusion bodies, and polykaryocyte formation. Microscopic examination of the corneal replicas shows the intranuclear lesions and rounding of cells up to about 2 mm away from the dendritic ulcers. These areas appear normal on clinical examination.


Histology of spheroidal degeneration of the cornea in Labrador. Nine specimens of the corneas of patients from Labrador and Northern Newfoundland affected by spheroidal degeneration (climatic droplet keratopathy) have been examined microscopically. Histochemical stains confirmed studies of similar corneal degenerations from other geographical areas that the droplets contain a protein which does not have all the characteristic properties of elastic tissue. Staining compatible in some instances with fibrin and "fibrinoid" was found. By immunoperoxidase techniques the droplets were located in the zones of greatest concentration of various plasma constituents, especially albumin and immunoglobulins G and A. Reasons are given why the abnormal deposits are not thought to be derived directly from corneal collagen. It is suggested that some of the plasma proteins, which are known to be diffusing through the cornea from the limbal vessels under normal conditions are acted upon by the ultraviolet light reflected from the ice of Labrador and degraded so that they accumulate in the superficial stroma.


Keratitis from abuse of corneal anaesthetics. Topically applied anaesthetics are potentially dangerous, as frequent and continuous application may lead to anaesthetic-induced keratitis. Three patients with serious corneal lesions are described.


An illustration of an in-vivo corneal response to a soft lens presoaked in a non-isotonic solution. The use of hydrophilic gel contact lenses as a carrier of a non-isotonic saline solution provokes an irreversible increase in corneal thickness in the in-vivo rabbit eye. Studies on humans confirm the nature of the animal response, and it is therefore suggested that particular care should be taken when patients prepare saline solutions.


Spectrum of optic nerve hypoplasia.

Spectrum of optic nerve hypoplasia. Optic nerve hypoplasia is a non-progressive condition characterised by subnormal vision and a subnormal number of optic nerve axons. It may be unilateral or bilateral, isolated or combined with other defects. Analysis of fundus photographs from a series of 7 patients with a stationary abnormality of different degrees showed that the functional defects could be closely correlated with defects in the retinal nerve fibre layer. Our observations show that the condition has a wide range of both functional and anatomical defects and that a subnormal diameter of the optic disc is not a requisite for the diagnosis. Presumably, there is also a wide variety of causes, not only a primary failure of development of retinal ganglion cells. We suggest that optic nerve hypoplasia can be viewed as a non-specific manifestation of damage to the visual system, sustained any time before its full development.


Simulation of energy metabolism in the simple-stomached animal. 1. A computer programme is described which simulates energy metabolism in the whole animal. Simulation was based on representation of the animal as a quasi-steady-state system. 2. Input for the programme consisted of the chemical composition of the diet and an estimate of either the maintenance energy requirement or an estimate of energy retention. 3. Simulation was performed by estimating the yield of adenosine triphosphate in the major metabolic pathways operative in simple-stomached animals, and on the utilization of adenosine triphosphate in major anabolic processes. 4. Results obtained from simulation were in close agreement with experimental observations reported by McCracken (1975).


Effects of diet on the protozoa population in permeable continuous cultures of rumen contents. 1. Responses of the protozoa population to the composition and the components of the diet were studied in permeable continuous cultures of rumen contents. 2. In Expt 1 a study was made of responses to diets containing different combinations of rice straw, lucerne meal and mixed concentrates given to the cow supplying inocula for cultures. It was found that (1) when a diet devoid of concentrates was fed, entodiniomorphs decreased in numbers; (2) their numbers increased with the increase in the amount of concentrates; (3) holtrich numbers were hardly affected by the type of diet; (4) rice straw and lucerne meal were not essential for protozoa to survive in culture medium. 3. In Expt 2 responses were determined to diets containing different combinations of maize, maize starch, sugars (glucose-sucrose (1:1, w/w)), soya-bean meal and isolated soya-bean protein. The results suggested the following relationship between dietary component and protozoa population. (1) The diet rich in both starch and sugars sustains the increased numbers of protozoa on the whole. (2) The diet rich in starch and deficient in sugars decreases selectively Dasytricha and small species of Entodinium such as Entodinium minimum and E. nanellum. The other species of protozoa, especially large entodiniomorphs such as Ophyroscolex and Polyplastron, are maintained at relatively high levels. (3) The diet deficient in starch and abundant in sugars decreases general entodiniomorphs except small species, while the numbers of Holotrichs are kept at relatively high levels.


The effect of frequency of feeding on the response by growing pigs to supplements of free lysine. 1. Six experiments, involving a total of 320 pigs, were conducted to examine the response of growing pigs to diets supplemented with free lysine when fed either once daily or in six equal portions at intervals of 3 h. The effect of dry or wet feeding on the response to free lysine was examined in Expt nos. 4 and 5. The assumption of linear lysine response over the range of lysine supplementation was examined in Expt no. 6. 2. A significant (P less than 0.01) interaction between lysine supplementation and frequency of feeding occurred for growth rate and food conversion ratio. Growth responses to the supplements of free lysine with once daily feeding were only 67% of that achieved with frequent feeding. 3. Wet feeding had no effect on the growth responses to free lysine supplements. 4. The lysine response was linear over the range of lysine supplementation examined. 5. The implication of these results to the interpretation of experiments examining the lysine requirements of pigs are discussed.


The assessment of zinc status of an animal from the uptake of 65Zn by the cells of whole blood in vitro. 1. 65Zn uptake by blood cells in vitro has been compared with plasma Zn concentration and plasma alkaline phosphatase (EC 3.1.3.1) activity as indicators of an animal's Zn status. 2. Dietary Zn deficiency, low food intake, reduced dietary protein content and endotoxin administration all reduced plasma Zn concentration in the rat. In each case there was a parallel reduction in plasma alkaline phosphatase activity and an increase in 65Zn uptake in vitro by cells of whole blood. 3. A similar relationship between the three measurements existed in sheep with lowered plasma Zn concentrations whether these were caused by dietary deficiency or by post-surgical stress. 4. 65Zn uptake by cells of whole blood did not differentiate dietary Zn deficiency from the other factors which reduce plasma Zn under 'field' conditions. 5. 65Zn uptake by the cells in whole blood in vitro was three to five times less rapid in blood of ruminant origin than in that from non-ruminants. This difference related to the erythrocytes rather than to the leukocytes or the plasma.


Effects of dietary molybdenum and sulphur on the distribution of copper in plasma and kidneys of sheep. 1. A 30-week study has been made in growing ewe lambs of the effects of dietary supplementation with molybdenum, or Mo plus inorganic sulphate on the distribution of copper in their blood plasma and kidneys. 2. The addition of 25 mg Mo and 5 g SO42-/kg diet increased Cu concentrations in plasma and kidney and decreased those in liver. Plasma caeruloplasmin activities (ferroxidase; EC 1.16.3.1) were unaffected. 3. Subcellular fractionation of the kidney cortex and gel filtration of the plasma and kidney cortex cytosol showed that the increased Cu content of these tissues was associated with abnormalities in the distribution of Cu. It appeared that both Cu and Mo were associated with the same proteins. 4. Dietary supplementation with Mo alone (25 mg/kg) had no effect on plasma or kidney Cu concentrations, suggesting that S metabolism is involved in the formation of the abnormal Cu-binding proteins in plasma and kidney. 5. The results are discussed in relation to the induction of Mo-induced Cu-deficiency states and the possible involvement of thiomolybdates.


Methane production in the rumen and lower gut of sheep given lucerne chaff: effect of level of intake. 1. Methane production rates were estimated simultaneously in the rumen and caecum of sheep given 200, 400, 600, 800 and 1000 g lucerne (Medicago sativa) chaff/d using isotope dilution techniques. Estimates were also made of volatile fatty acid (VFA) production in the rumen at each level of feeding. In all studies three to four animals were used at each level of intake. 2. Production of VFA and of methane were both related to digestible energy (DE) intake. Regression lines for both VFA production and methane production v. DE intake had significant intercepts indicating an input of endogenous, fermentable organic matter into the rumen in excess of 50 g/d. 3. The values obtained for rates of methane production were compared with those calculated from stoicheiometric equations relating rates of methane and VFA production. Comparisons of methane production with that predicted from DE intake were also made. 4. Balances for digestion of food determined for the rumen indicated that the energies in the end-products were more than 100% of the DE intakes of lucerne chaff. Correction for fermentation of apparent endogenous materials resulted in more realistic values. Endogenous materials appeared to make a significant contribution to VFA and methane production, particularly at low levels of intake.


The effect of litter size on placental blood flow and placental calcium transfer in the multifoetate guinea-pig. 1. Placental blood flow rate and calcium transfer rate were measured at 61 d of pregnancy in guinea-pigs carrying between one and eight foetuses. 2. Placental blood flow rate was significantly correlated with foetal weight. Ca transfer rate was related to placental size. Irrespective of litter size the mean amount of Ca transferred across a placenta was between 0.22 and 0.34 mg/h per g placental tissue. 3. It was concluded that there was a limit to the rate of transfer which was produced by a combination of limitations in placental blood flow rate, maternal plasma Ca concentration and placental tissue transfer capacity.


Prospective comparison of modified fat-high-carbohydrate with standard low-carbohydrate dietary advice in the treatment of diabetes: one year follow-up study. 1. A prospective randomized study of two dietary regimens has been started in newly-diagnosed diabetics to determine their effect on circulating metabolites and on diabetic complications. 2. During the first year of treatment the fasting plasma glucose concentrations on both the low-carbohydrate diet and the high-carbohydrate, modified-fat (MF) diet showed a similar decrease. 3. Plasma cholesterol showed a sustained decrease only in patients recommended a MF diet. Transient changes in plasma triglyceride concentrations occurred in patients on both dietary regimens. 4. Increased plasma cholesterol levels are associated with atheromatous disease which is common in diabetics in Europe and North America. A MF diet may therefore have an advantage in that it lowers the plasma cholesterol as well as being effective in lowering the plasma glucose.


Trace nutrients. Selenium in British food. 1. The amount of selenium in nationally representative samples of prepared and cooked groups of foods, and in a variety of raw individual foods, was determined fluorimetrically. 2. The average British diet was calculated to provide approximately 60 microgram Se/d, of which half was derived from cereals and cereal products and another 40% from meat and fish. Milk, table fats, fruit and vegetables provided little or no Se. 3. Individual foods which were particularly rich in Se (greater than 0.2 mg/kg) included 'bread-making' and wholemeal flours, kidney, fatty fish, brazil nuts (Bertholletia excelsa) and several other varieties of nut. In contrast, breast milk and other foods for babies (except some cereal products) contained little Se. 4. The total intake, and the amounts of Se in major foods, were lower than in most other studies. This is probably the result of the comparatively low levels of this element in British soil.


The development of obesity in preweaning obob mice. 1. The body compositions of obob and lean (ob+ and ++) mice at 10, 12, 17 and 28 d of age were investigated using a 'cold stress' test to identify the two groups. 2. At each of these ages the obob mice were found to contain significantly more fat than the lean. At 10 d 20% more fat was present and by 17 d the increase was 72%. The obob mice at 28 d contained nearly three times as much fat as the lean. 3. Carcass energy was significantly higher in obob mice at all ages investigated. 4. Other changes in body composition found in the 28 d obob mice, i.e. a reduction in total carcass nitrogen and water content, were already established in the 17-d-old mice but differences at 10 and 12 d were not apparent. 5. The livers of obob mice were significantly heavier than those from lean control mice at 28 d but no differences were detected at the earlier ages. 6. The results are discussed with reference to the early origin of obesity in obob mice.


Fat-free body mass from skinfold thickness: a close relationship with total body nitrogen. Fat-free body mass is an important component of body composition which is of particular interest to nutritionists and related workers. Fat-free mass has been determined from the simple measurement of skinfold thickness and it has been demonstrated that there is a close correlation with total body nitrogen even though the test subjects had a wide range of nutritional status.


The energy content of rat carcasses prepared for analysis by different methods. The effect of oven-drying on the gross energy content of rat carcasses was investigated. No losses of carcass energy could be ascribed to oven-drying per se.


A comparison of the effects of dietary calcium and phosphorus deficiency on the in vitro and in vivo metabolism of 25-hydroxycholecalciferol in the chick. 1. Young chicks fed a diet deficient in calcium showed an eightfold increase in the in vitro renal production of 1,25-dihydroxycholecalciferol (1,25-DHCC) and those fed a diet deficient in phosphorus showed a threefold increase when compared to chicks on a normal diet. 2. The in vivo accumulation of 1,25-DHCC in the gut mucosa was doubled in both low-Ca and low-P groups as was the rate of Ca absorption from the duodenum and the Ca-binding protein activity. The accumulation of 1,25-DHCC in bone increased threefold in the low-Ca group but showed no change in the low-P group. 3. It was concluded that the increased rate of Ca absorption found in dietary P deficiency depends rather upon the capacity of the gut mucosa to accumulate larger amounts of 1,25-DHCC than upon an increased renal production of this metabolite. The mechanism by which this is achieved is unknown, but it unlikely to be a general increase in availability of 1,25-DHCC since no rise occurred in bone 1,25-DHCC levels.


Proton nuclear magnetic resonance studies of hemoglobin Providence (beta82EF6 Lys replaced by Asn or Asp): a residue involved in anion binding. High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.


Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin. High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.


Covalent structure of a group-specific protease from rat small intestine. Appendix: crystallographic data for a group specific protease from rat intestine. "Group-specific" protease (GSP) is a serine protease, obtained from rat small intestine, which preferentially inactivates the apo forms of certain pyridoxal phosphate requiring enzymes. The enzyme contains 224 amino acid residues in a single polypeptide chain and three disulfide bonds. In the present work the covalent structure has been determined and its homologous relationship to those of chymotrypsin, trypsin, and elastase has been established (approximately 33% identity with each). The residues forming the "charge-relay" system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in GSP, whereas an alanyl residue at position 176 of GSP corresponds to a residue which participates in the primary substrate binding site in serine proteases (Asp-177 in trypsin; Ser-189 in chymotrypsin). Three disulfide bonds in GSP occur in similar positions in chymotrypsin, trypsin, and elastase. However, GSP lacks a disulfide bond which is present in all known serine proteases (linking Cys-191 to Cys-220 in chymotrypsin). In view of the close proximity of this bond to both the primary and the antiparallel binding sites of various serine proteases, it is likely that its absence in GSP is related to the substrate specificity of this enzyme. It is concluded that GSP diverged from a common ancestor preceding chymotrypsin but following trypsin.


Surface areas of lipid membranes.

Surface areas of lipid membranes. Upon photolysis, alkyl pentacyanocobaltate complexes generate alkyl radicals which react rapidly and specifically with nitroxide radicals, and which do not penetrate phospholipid bilayers. By measuring the loss of paramagnetic resonance signal intensity when multilamellar liposomes containing a small amount of spin-labeled lipid are exposed to these radicals, we have measured the proportion of lipid on the external surface of liposomes. We have shown that liposomes prepared under specified conditions from dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and binary mixtures of dipalmitoylphosphatidylcholine and cholesterol all have the same proportion of external lipid.


Multiple thymine dimer excising nuclease activities in extracts of human KB cells. Crude extracts of human KB cells grown in suspension culture contain enzyme activity that catalyzes the preferential excision of thymine-containing pyrimidine dimers from UV-irradiated E. coli DNA specifically incised adjacent to dimer sites. Fractionation of KB cell crude extracts reveals the presence of three such activities with distinct affinities for both DEAE-cellulose and phosphocellulose. One of the activities (activity B) is distinguished by its s 20,w (2.6) and isoelectric point (9.0) from the other two (activities A and C) which have similar s 20,w's (3.0-3.2) and isoelectric points (6.0). All three differ in their extent of stimulation by divalent cation and inhibition by NaCl or a sulfhydryl group inhibitor. These results indicate that multiple 5' leads to 3' dimer excision nuclease  activities exist in human cells; however, there is as yet no direct evidence that these enzymes are functional in nucleotide excision repair in vivo.


Mode of chromatin reconstitution. Elements controlling globin gene transcription. The mode of reconstitution has been determined for the factors that control globin gene transcription in chicken reticulocyte chromatin. During chromatin reconstitution, elements in the nonhistone protein fraction that control the globin gene reassociate with the DNA before the histones. From our results, it appears that it is necessary for these elements to reassociate with the DNA before the histones for the globin gene to be transcribed.


S-trifluoroacetonyl-coenzyme A:a 19F analogue of acetyl-coenzyme A. S-Trifluoroacetonyl-coenzyme A has been synthesized in 87% yield by reaction of 1,1,1-trifluoro-3-bromopropanone with trilithium coenzyme A in presence of pyridine. The compound was characterized by its ultraviolet absorption spectrum and 1H and 19F nuclear magnetic resonance spectra. The alpha-methylene protons of the S-trifluoroacetonyl group exchanged with D2O and showed a pKa of 9.85 in S-trifluoroacetonylmercaptoethanol. S-Trifluoroacetonyl-coenzyme A is a competitive inhibitor of porcine heart citrate synthetase (Ki = 0.16 mM). It forms a binary complex with the enzyme and a ternary complex with enzyme/oxaloaetate binary complex, as evidenced ty the 19F shift. S-Trifluoracetonyl-coenzyme A and S-trifluoroacetonylmercaptoethanol form weak to moderately strong complexes with alpha-cyclodextrin and show little or no interaction with the methylglucose polysaccharide and lipopolysaccharides from Mycobacterium smegmatis [Smith, W. L., & Ballou, C. E. (1973) J. Biol. Chem. 248, 7118]. S-Trifluoroacetonylmercaptoethanol probably forms an inclusion complex with alpha-cyclodextrin because the interaction is reversed by compounds that do form inclusion complexes.


Phase transitions of the purple membranes of Halobacterium halobium. Purple membranes of Halobacterium halobium were studied by differential scanning calorimetry. No transition was detected at temperatures below 70 degrees C. A small endothermic transition was seen at about 80 degrees C and a larger one at 100 degrees C. The larger transition is the irreversible denaturation of bacteriorhodopsin. The smaller transition is accompanied by a change in the visible absorption spectrum and is believed to be reversible, involving a cooperative change in crystalline structure of the membrane.


Mechanism of inactivation of ornithine decarboxylase by alpha-methylornithine. Ornithine decarboxylase from Lactobacillus 30a is gradually inactivated by treatment with alpha-methylornithine, but activity is restored by treatment of the inactivated enzyme with pyridoxal phosphate. Inactivation of the enzyme is associated with formation of pyridoxamine phosphate and 5-amino-2-pentanone, alpha-Methylornithine is decarboxylated by the enzyme about 6000 times more slowly than is ornithine under the same conditions. These observations provide an explanation for the previously observed inhibition of ornithine decarboxylase by alpha-methylornithine [M. M. Adbel-Monem, N. E. Newton, and C. E. Weeks (1974), J. Med. Chem. 17, 4447]: alpha-Methylornithine undergoes a decarboxylation-dependent transamination as a result of incorrect protonation of the quinoid intermediate which is formed by decarboxylation of the enzyme-bound pyridoxal phosphate-substrate Schiff base. This protonation produces inactive enzyme. Decarboxylation of ornithine by this enzyme produces a small amount of 4-aminobutanal, presumably also by decarboxylation-dependent transamination.


Triplet state of tryptophan in proteins: the nature of the optically detected magnetic resonance lines. Optical detection of magnetic resonance (ODMR) has been employed to examine the homogeneity of the tryptophan environment, both of the isolated residue in solvent, and of tryptophan in glucagon and lysozyme and azurin B (Pseudomonas aeruginosa). From the shifts in the zero-field splittings, we can safely conclude that tryptophan in lysozyme, azurin B, or glucagon does not have the same type of solvent interaction as the free residue. However, by "burning holes" in the OSMR lines, it is evident that the lines in these cases are inhomogeneously broadened. From the relative line widths and hole widths, it appears that ODMR can be used to examine the relative diversity of interactions for a luminescent amino acid in a protein. We have followed the ODMR line characteristics in a progression from free N-acetyl-L-tryptophanamide, to tryptophan in lysozyme, to "denatured" lysozyme, and present evidence that the line widths narrow as the tryptophan residues become less solvent accessible.


Primary structure of the lambda repressor. The complete covalent structure of the bacteriophage lambda repressor has been determined by sequential Edman degradation, gas chromatographic-mass spectrometric peptide sequencing, and DNA sequencing of the repressor gene cI. The repressor is a single-chain, acidic protein containing 236 amino acids. The amino terminal 40 residues are highly polar and basic. Lysines and arginines in the sequence tend to be clustered.


Nuclear magnetic resonance study of fibrinogen-like peptides and their structure in dimethyl sulfoxide and water. The 1H nuclear magnetic resonance (NMR) spectra of four fibrinogen-like oligopeptides (H-Gly-Pro-Ala-NH2, H-Arg-Gly-Pro-Ala-NH2, H-Val-Arg-Gly-Pro-Ala-NH2, and H-Gly-Val-Arg-Gly-Pro-Ala-NH2) in dimethyl-d6 sulfoxide (Me2SO-d6), and of the hexapeptide in water, and the 13C NMR spectrum of H-Gly-Pro-Ala-NH2 in Me2SO-d6, were recorded and interpreted in terms of preferred conformations in solution. Each peptide exists in Me2SO-d6 as in a 30:70 mixture of cis and trans isomers about the Gly-Pro bond, and the hexapeptide in water is solely the trans isomer. For the trans isomers in Me2SO-d6, there is a hydrogen bond between the Gly CO group and one of the C-terminal primary amide hydrogens, and a beta turn involving the Gly-Pro-Ala-NH, section of the molecules. A strong NOE between Pro CalphaH and Ala NH for the trans isomer of the tripeptide in Me2SO-d6 completes the identification of this structural feature as a type II beta turn.


Effects of shearing on chromatin structure. The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.


Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates. HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM  to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.


Characterization of DNA binding protein from rat liver chromatin which decreases during growth. A nuclear nonhistone protein which decreases in chromatin during growth (Yeoman, L. C., et al. (1975) Cancer Res. 35, 1249) has been isolated in high purity from the chromatin of normal rat liver nuclei by gel electrophoresis and column chromatography. This protein, designated BA (Yeoman, L.C., et al. (1973) Biochem Biophys. Res. Commun. 53, 1067), has a molecular weight of 31 000, an acidic to basic amino acid composition ratio of 0.9, and contains one tryptophan residue per molecule. Hydrazinolysis indicated protein BA has a lysine carboxyl terminus; however, the amino terminal is blocked as no reaction occurred with dansyl chloride. Maps of tryptic peptides of protein BA contained 46 spots. Protein BA binding to various DNAs was examined by the nitrocellulose filter assay. Binding was slightly enhanced by 2mM Mn2+ion; Mg2+, however, decreased binding. Binding was optimal at neutral pH and an ionic strength of 0.2 M [NaCl]. Equilibrium competition binding studies indicated a binding preference of protein BA for dA-dT rich DNA.


Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilorostris. The bioluminescent reaction of Oplophorus takes place when the oxidation of coelenterazine (the luciferin) with molecular oxygen is catalyzed by Oplophorus luciferase, resulting in light of maximum intensity at 462 nm and the products CO2 and coelenteramide. Oplophorus luciferase has now been obtained in a highly purified state. Optimum luminescence occurs at pH 9 in the presence of 0.05--0.1 M NaCl at 40 degrees C, and, due to the unusual resistance of this enzyme to heat, visible luminescence occurs at temperatures above 70 degrees C when partially purified enzyme is used. The specific activity of purest preparations is 1.75 X 10(15) photons s-1 mg-1 at 23 degrees C. At pH 8.7, native luciferase has a molecular weight of approximately 130 000, apparently comprising 4 monomers of 31 000; at lower pHs, the native luciferase tends to polymerize. The quantum yield of coelenterazine is 0.34 at 22 degrees C with this enzyme. After the luminescent reaction, the spent solution is nonfluorescent, and likewise solutions of luciferase alone. When the bioluminescent reaction was carried out in the presence of 18O2, the product CO2 contained more than 50% C18O16O, supporting the dioxetane mechanism, but without ruling out the linear peroxide mechanism.


Mitochondrial framework (reticulum mitochondriale) in rat diaphragm muscle. Reconstitution of rat diaphragm mitochondria has been carried out with the use of the serial section technique. It is shown that mitochondrial material is organized as networks transpiercing the I band regions of the muscle near the Z-discs. Each network forms tubules, oriented perpendicular to its plane, and branches, connecting the network with mitochondrial clusters in the fiber periphery. Such a system, defined as mitochondrial reticulum, is found to be characteristic of the diaphragm of adult animals. It is absent in the diaphragm of rat embryos and newborn rats. The junctions of the branches of mitochondrial reticulum are described. In the junction site, the outer membranes of two mitochondrial branches are in contact, and spaces between outer and inner membranes are filled with an osmiophilic substance. No junctions were found in the embryos and in newborn animals whose diaphragm contains single, elliptical or worm-like mitochondria. The hypothesis is put forward that the mitochondrial reticulum serves as a system for transport of energy, oxygen and fatty acid residues along mitochondrial membranes over distances commensurable with the muscle fiber diameter.


The effects of uncoupler on the rates of cytochrome oxidation and reduction in the photosynthetic bacterium, Chromatium. Evidence for a possible cytochrome switching. The uncoupler, m-chlorocarbonyl cyanide phenylhydrazone (CCCP) added to anerobic, dark-adapted whole cells of Chromatium vinosum is found to speed the reduction of cytochrome after oxidation by laser or by steady illumination and, subject to unknown factors, to slow the laser-induced oxidation. There is considerable evidence, including spectra and loss of low-temperature oxidizability that this results from a switch of the identity of the cytochrome oxidized from the low-potential cytochrome c-552 to the high-potential cytochrome c555. Redox control and/or control by conformational movements of the cytochromes, either being coupled to energy transduction in the cyclic system, are suggested as mechanisms for the switching. If the switching hypothesis is not accepted, the increased rate of reduction could alternatively be explained by postulating a phosphorylation site in the reduction pathway.


Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa. Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 +/- 20 mM--1 . CM--1. The molecular weight based on protein and chromophore assays was found to be 1.5 . 10(5); the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.


Isolation and characterization of light harvesting bacteriochlorophyll.protein complexes from Rhodopseudomonas capsulata. The isolation of two native light harvesting bacteriochlorophyl.protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant A1a+ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight. Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.


Absorption and circular dichroism spectra of chloroplast membrane fragments from spinach, barley and a barley mutant at room temperature and liquid nitrogen temperature. The absorption and CD spectra of chloroplast fragments from spinach, barley and a barley mutant (chlorophyll b-minus) were studied at temperatures of 23 degrees C and --196 degrees C. The CD spectrum of wild type barley and spinach at --196 degrees C showed troughs at 640, 653, 676 and 695 nm and a maximum at 667 nm. The CD spectrum of the barley mutant at --196 degrees C consisted of a large trough at 684 nm, a small trough at 695 nm and a positive peak at 670 nm. A new feature observed at --196 degrees C but not at 23 degrees C is the trough at 640 nm. This 640 nm CD signal is missing in the CD spectrum of the barley mutant. It is attributable to the light-harvesting chlorophyll a/b protein which appears to be missing in the mutant. Another new feature, the trough at 695 nm, was observed in the CD spectra of spinach, barley and the barley mutant at --196 degrees C. The 695 nm trough appears to be sensitive to detergents and it may be due to a labile chlorophyll a.protein complex. Possible interpretations of the data are discussed.


The modulation of membrane fluidity by hydrogenation processes. II. Homogeneous catalysis and model biomembranes. A homogeneous catalyst, chlorotris (triphenylphosphine) rhodium (I) has been incorporated into model biomembrane structures in the form of lipid bilayer dispersions in water. This enables the hydrogenation of the double bonds of the unsaturated lipids within the bilayers to be accomplished. To decide the optimum conditions for efficient hydrogenation the reaction conditions have been varied. The effect of catalyst concentration, hydrogen gas pressure and lipid composition (with and without cholesterol) have all been studied. The partition of the catalyst into the lipid medium was checked by rhodium analysis. The results show that an increase of catalyst concentration or an increase of hydrogen gas pressure leads to increasing rates of hydrogenation. Successful hydrogenation was accomplished with different types of lipid dispersions (mitochondrial, microsomal and erythrocyte lipids). A selectivity of the homogeneous hydrogenation process is indicated. The polyunsaturated fatty acyl residues are hydrogenated at an earlier stage and at a faster rate than the monoenoic acids. Furthermore, an increase in the proportion of cholesterol to lipid within the bilayer structures causes a progressive decrease in the rate of hydrogenation. The fluidity of the lipid bilayers can be altered to such an extent by the hydrogenation process that new sharp endotherms corresponding to the order-disorder transition of saturated lipids occur at temperatures as high as 319 K. Some potential uses of hydrogenation for the modulation of cell membrane fluidity are discussed as well as the design of new types of catalyst molecules.


Interpretation of light scattering associated with prolonged neural activity and temperature changes. Changes in light scattering from lobster giant axon which accompany the action potential were observed during periods of prolonged stimulation and as a function of temperature. At an initial temperature of 10 degrees C most (more than 90%) axons produced positive light scattering signals which increased in amplitude when the temperature was lowered. At 2 and 5 degrees C approximately half of the axons produced positive scattering signals. The remaining half produced negative scattering signals which became positive when the temperature was raised to 10 degrees C. The amplitude of the negative signals followed sigmoid transition to positive values as a function of time. The time and temperature dependence of the signal are interpreted in terms of differential changes between the indices of refraction of the membrane matrix and the open or closed early activation channel.


Preparation of liver microsomes with high recovery of endoplasmic reticulum and a low grade of contamination. A modified procedure for preparing the microsomal fraction from rat liver was developed with the aim of increasing the recovery without increasing the degree of contamination. 87% of the membranes of the microsomal fraction isolated from the first mitochondrial (10 000 X g) supernatant originates from the endoplasmic reticulum, representing a 35% yield. By gentle resuspension of the 10 000 X g pellet followed by differential centrifugation a second crop of microsomes can be prepared which, together with the first crop, gives a 55% total recovery of microsomal markers. 87% of the protein in this second crop also originates from the endoplasmic reticulum and this fraction has properties similar to those of the first crop. Contaminating membranes include Golgi membranes (0.6% of the total protein), mitochondria (2.5%), lysosomes (5%) and plasma membranes (5%). Collecting further crops increases the contamination. Subfractionation studies revealed almost identical distributions of ribosome-rich, ribosome-poor and smooth membranes in the two crops of microsomal fractions. The results obtained after treatment of the animals with phenobarbital or methylcholantrene were similar to those obtained with control animals; but in the case of methylcholantrene treatment the second crop represents a larger portion of the total membranes of the endoplasmic reticulum.


Fluorescence measurements of environmental relaxation at the lipid-water interface region of bilayer membranes. Nanosecond time-resolved emission spectroscopy is used to characterize the complex fluorescence behavior of the probe 2-p-toluidinonaphthalene 6-sulfonate (2,6 p-TNS) when adsorbed to several bilayer membrane system. These include egg phosphatidylcholine vesicles with and without added cholesterol as well as erythrocyte ghost membranes. In each case a nanosecond time-dependent shift of the fluorescence emission to lower energy follows pulsed photoexcitation. The properties of the time-resolved surfaces obtained are consistent with a non-exponential decay law which describes a continuous interaction process of 2,6 p-TNS with its local environment in the membrane. This environment consists in part of polar residues (water plus polar head region) undergoing nanosecond motions. The pure phosphatidylcholine bilayer system was studied at four temperatures and electronic and spectral relaxation contributions to the total fluorescence decay were separated. Temperature coefficients for empirical rate parameters derived for the separated processes were obtained. It appears that a treatment of the fluorescence behavior of amphiphilic probes such as 2,6 p-TNS adsorbed to bilayer membranes at temperatures near ambient in which a single lifetime and radiative decay channel have been assumed is inappropriate.


Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins. Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.


Protein-mediated transbilayer movement of lysophosphatidylcholine in glycophorin-containing vesicles. 1. Sonicated glycophorin-containing vesicles of dioleoyl phosphatidylcholine have been made. The outside-inside distributions of the lipid molecules in these vesicles was measured with NMR and was found to be comparable with that of protein-free vesicles. 2. The transbilayer distribution of palmitoyl lysophosphatidylcholine in these vesicles is such that they have a significantly higher content of the lyso-compound in the inner monolayer when compared with vesicles without glycophorin. 3. Lysophosphatidylcholine, added to pre-existing glycophorin-containing vesicles, is incorporated in the outer monolayer of these vesicles. Subsequently it is able to move to the inner monolayer with an estimated half time of about 1.5 h at 4 degrees C. This was measured with 13C-NMR using [N-13CH3]lysophosphatidylcholine. 4. Treatment of co-sonicated vesicles of phosphatidylcholine and lysophosphatidylcholine containing glycophorin with the enzyme lysophospholipase results in a complete degradation of the lyso-compound. A half time of transbilayer movement of lysophosphatidylcholine during this experiment was estimated to be about 1 h at 37 degrees C.


The complexing of guanine nucleosides by poly(inosinic acid). Under conditions where it is single-stranded (pH 7.0, 0.05 M Na+), poly(inosinic acid) complexes guanosine and 2'-deoxyguanosine in a strongly cooperative process that ultimately results in gel formation. The binding isotherm for formation of the poly(I).deoxyguanosine complex at 3.5 degrees C shows that it contains approximately one molecule of deoxyguanosine bound per IMP residue of the poly(I). This is compatible with the complex having a structure similar to that of the four-stranded secondary structure of poly(I) in which two of the poly(I) strands are replaced by helical arrays of deoxyguanosine molecules. The complex is laevorotatory at wavelengths longer than 300 nm. Optical rotation and ultraviolet absorbance versus temperature profiles show that on thermal dissociation the complex undergoes a co-operative helix-coil transition with Tm in the range 10--20 degrees C.


Regulation of hepatic synthesis of proteins by the chronology of protein ingestion. Circadian variations in liver protein synthesis were were assessed in control rats fed a mixed 10% protein diet and in rats fed proteins as a separate meal either at 09:00 (SF 09) or at 21:00 (SF 21) and provided with a protein-free diet ad libitum. Protein synthesis was measured by incorporation of labelled leucine over a short period of time (15 min) at time-points regularly spaced over 24 h. In controls, the circadian variations observed were of moderate amplitude (from 2.75 mg/h per g at 09:00 to 5.77 mg/h per g at 06:00) correlated with increased protein and RNA contents of the liver. In separately fed animals ingestion of the protein meal triggered a 300% increase in protein synthesis within 1 h while the feeding pattern was unaltered. In the SF 09 group, high synthetic activity was not followed by an increase of hepatic protein content while hepatic urea concentrations were sharply increased and glucogenic amino acid pools were greatly depleted. It is suggested that the high influx of amino acids consecutive to the absorption of the dietary proteins is the key factor stimulating protein synthesis, while synchronisation with the energetic metabolism controls the degree of degradation. The possible involvement of variations in the insulin to glucagon ratio is discussed.


The activity of mammalian polynucleotide ligase on x-irradiated DNAs. Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.


Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver. tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA(N2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1-)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNAMetf was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNAMetf is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the Km for S-adenosylmethionine is 3 micrometer and Ki for S-adenosylhomocysteine is 0.11 micrometer for (guanine-1-)-methyltransferase. (N2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNAPhe, tRNAVal and tRNAArg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5'-terminus of these tRNAs. The methylation of tRNAPhe is maximally stimulated by 100 micrometer spermidine with a pH optimum of 8.0. (N2-Guanine-)-methyltransferase has a Km for S-adenosylmethionine of 2 micrometer and a Ki for S-adenosylhomocysteine of 23 micrometer with E. coli K-12 tRNA as methyl acceptor.


A conformational basis for the antiviral inactivity of tetrazole ribonucleosides. The antiviral activity of ribavirin has been associated with its inhibition of the enzyme, IMP dehydrogenase. The ability of ribavirin to inhibit this enzyme has previously been shown to be related to its stability in the high anti glycosidic conformation. The antiviral effectiveness of several analogs of ribavirin have been investigated recently. The evidence indicates their antiviral effectiveness is related to their stability in the high anti conformation. Recently the disposition of purine analogs that pass through the inosine monophosphate branch point has been investigated. The results of these studies are consistent with the concept that the conversion of IMP to XMP requires the high anti conformation and that the conversion of IMP to adenylosuccinate requires some other conformation, possibly the anti conformation.


Complexes of DNA with arginine-rich and slightly lysine-rich histones. Transcription and electron microscopy. Calf thymus DNA was reconstituted with arginine-rich histones H3 and H4 and slightly lysine-rich histones H2A and H2B respectively. Complexes containing histones H3 and H4 exhibit nucleosome-like structures when examined in the electron microscope and show a restriction of in vitro transcription similar to that obtained for reconstitutes made up from the four histones H2A, H2B, H3 and H4. One the contrary, complexes of DNA and histones H2A and H2B create different morphological structures of short stretches of bound histones and do not cause a template restriction in vitro.


DNA damage during the peroxidase-catalyzed aerobic oxidation of isobutanal. The aerobic oxidation of isobutanal catalyzed by peroxidase, when carried out in the presence of DNA, produces alkali-sensitive bonds in this macromolecule. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Since triplet acetone has been recently identified as an intermediate in this oxidation (Durán, N., Faria Oliviera, O.M.M., Haun, M. and Cilento, G. (1977) J. Chem. Soc. Chem. Commun., 442--443), this species is a likely candidate for the entity which brings about the lesions, via transfer of its electronic energy to DNA.


Ribonuclease H activity in cultured plant cells. Ribonuclease H (RNAase H) was extracted from cultured plant cells, strain GD-2 and characterized. RNAase H activity in logarithmical growing cells is much higher than that of stationary cells, and the response of RNAase H activity was very similar to that of DNA polymerase after culture. The activities of RNAase, DNAase, phosphodiesterase and alkaline phosphatase decrease parallel with the increase in growth, and increase to stationary phase, contrasting with those of DNA polymerase and RNAase H.


Comparative utilization of bromodeoxyuridine and iododeoxyuridine triphosphates for mammalian DNA replication in vitro. Relative utilization, of dThd, BrdUrd, and IdUrd as exogenous precursors of DNA in vivo was compared with utilization of the respective triphosphates in vitro. To measure relative utilization, exponentially growing P-815 or CHO cells were incubated with different mixtures of dThd/BrdUrd or dThd/IdUrd in the presence of FdUrd and dCyd, and Brij-58 lysed cells with mixtures of dTTP/BrdUTP or dTTP/IdUTP. The degree of substitution of dTMP by BrdUMP or IdUMP in DNA newly synthesized in vivo or in vitro was determined by isopycnic centrifugation. BrdUrd was utilized by intact cells with equal efficiency as dThd, while IdUrd was less efficiently incorporated when the total concentration of dThd and the tested analogue was 3.10(-5) M. During semiconservative DNA replication in vitro, however, dTTP, BrdUTP and IdUTP were utilized with equal efficiency. Thus, we conclude that observed preference for dThd by intact cells incubated with dThd/IdUrd mixtures is related to characteristics of precursor metabolism and not to different affinities of the replication complex for the respective precursors. dUTP was rapidly dephosphorylated in vitro and no incorporation into DNA was detected.


Fractionation of renatured repetitive human DNA according to thermal stability, sequence length, and renaturation rate. Renatured repetitive human DNA sequences have been fractionated according to three criteria: length, thermal stability, and rate of renaturation. Satellite sequences are found in the long repetitive sequence fractions and renatured satellite sequences are well base paired by the criterion of thermal stability. No satellites are observed in short repeated sequences. Short repeated sequences are interspersed with single copy sequences and are poorly base paired. This implies satellite components are relatively homogeneous in base sequence whereas short interspersed repeated sequences are inexact copies of each other. A method for isolating satellite DNAs based on their high thermal stabilities is discussed.


Nuclear columns, Kinetics of RNA synthesis and release in isolated rat liver nuclei. By continuous perfusion of columns containing isolated immobilized rat liver nuclei with media containing labeled RNA precursors, the in vitro synthesis and release of RNA was studied. The combined reaction of synthesis and release could be adjusted to proceed at a constant rate. The reaction rate responded to variation of termperature, ionic conditions, nucleoside triphosphate concentration and to the addition of RNA polymerase inhibitors. During 60 min perfusion approximately equal amounts of radioactive low molecular weight RNA and of ribonucleoproteins were released. Pulse-chase experiments showed that the low molecular weight RNA was synthesized throughout the perfusion and released immediately after formation. The ribonucleoproteins were primarly labeled during the first period of perfusion and were gradually released. Synthesis of RNA contained in the ribonucleoproteins was inhibited by low alpha-amanitin concentrations, indicating that it was catalyzed by RNA polymerase II. The in vitro labeled ribonucleoproteins exhibited properties of the stable nuclear particles which can be extracted from isolated nuclei after rapid in vivo labeling of RNA. They had a buoyant density of 1.41--1.43 in CsCl, were partially unstable in 1% deoxycholate, but stable in 0.1% deoxycholate, in 100 mM NaCl and in 10 mM EDTA. Due to the dilution by the perfusion medium, the ribonucleoproteins sedimented with a peak at 22--27 S, and not at 30--45 S. The RNA synthesized in the immobilized nuclei was not degraded during the perfusion. Less than 20% was gradually released, whereby the 20--30 S peak zone was reduced. While the properties of the in vitro labeled ribonucleoproteins and of rapidly in vivo labeled ribonucleoproteins were the same, the kinetics of their release differed.


5-Substituents in the uridine moiety and their effect on the conformation of ApU-type dinucleoside phosphates. The ApU analogues ApT, Apcl5U, Apbr5U, Apa5U and Apno5(2)U were synthesized with the aid of ribonuclease U2 starting from 2',3'-cyclic Ap and the respective uridine derivatives. For these compounds the ultraviolet data, the difference spectra, the hypochromism and the temperature dependence of the CD spectra are reported. The dimerisation shifts of the pyrimidine protons which were obtained from the 100 MHz PMR spectra confirm the optical results. The influence of the substituents in the 5 position of the uracil ring on base-base interaction and the conformation of the dinucleoside phosphates is discussed with respect to the van der Waals radii and the electronic effects of these groups. As calculated from the hypochromism the dinucleoside phosphates can be arranged according to decreasing base-base interaction: Apno5(2)U greater than Apbr5U approximately ApT greater than Apcl5U greater than ApU greater than Apa5U.


The role of nonhistone chromosomal proteins in the interaction of prostate chromatin with androgen.receptor complex. Rat prostate nonhistone chromatin proteins were fractionated by sequential extraction with 0.35 M NaCl and 2.0 M NaCl into 0.35 M NaCl-soluble, 2 M NaCl-soluble and -insoluble (residual) fractions. Interactions of [3H]dihydrotestosterone.receptor complex with reconstituted chromatin lacking one of these fractions and with reconstituted hybrid chromatin using spleen fractions indicate that binding of chromatin to androgen.receptor is largely determined by the residual fraction. The residual fraction of prostate also interacts directly with [3H]dihydrotestosterone.receptor complex target tissue-specifically similar to prostate chromatin. The DNA in the residual fraction is mostly bound by nonhistone proteins and the template activity of te residual DNA in transcription is severely restricted. These characteristics are discussed in relation to the acceptor role in the residual fraction in androgen action.


Identification of the methylated ribosomal proteins in HeLa cells and the fluctuation of methylation during the cell cycle. Methylated proteins from HeLa cell cytoplasmic ribosomes have been identified. At least seven proteins are methylated and four of them are mildly acidic. The nature of the methylated amino acid in each protein is presented. In synchronized HeLa cell culture, the extent of methylation for both subunits varies with the cell cycle. Methylation of the 40 S subunit occurs heavily in the late G1 phase whereas methylation of the 60 S subunit is most pronounced in the early S phase.


Supercooled water as medium for enzyme reactions at subzero temperatures. A water-in-oil emulsion technique was employed to investigate enzyme-catalyzed reactions at sub-zero temperatures in the supercooled liquid state. The results obtained with a monooxygenase (bacterial cytochrome P-450) clearly indicate the potentialities as well as the technical problems of the procedure which might be successfully used to investigate enzyme systems sensitive to cosolvents.


Purification and properties of Arthrobacter neuraminidase. Neuraminidase (EC 3.2.1.18) from an Arthrobacter species was purified homogeneity by conventional procedures (yield approx. 1 mg/1) and was judged to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Gel electrofocusing of neuraminidase revealed 1 major band (85-90%), pI 5.35 +/- 0.05, and 6 minor bands, whose pI ranged from 5.25 to 5.70, and each of which had catalytic activity. Arthrobacter neuraminidase is a monomeric glycoprotein of molecular weight 88 000, has an apparent Km of 7.8-10(-4) M for N-acetylneuraminlactose, is insensitive to inhibition by N-acetylneuraminic acid, and is about 2% carbohydrate by weight. The amino acid composition as well as the galactosamine and glucosamine content was determined. The enzyme can hydrolyze (alpha, 2-3), (alpha, 2-6), (alpha, 2-8) linkages. The active size of the enzyme appears to be inaccessible since no inhibition was observed by reagents known to modify sulfhydryl, lysyl, carboxyl, histidinyl, and argininyl residues. In contrast, N-bromosuccinimide at a 60-fold molar ratio to enzyme, gave complete inhibition. These results suggest that a tryptophan residue is essential for catalysis.


Purification and properties of three proteases from the larvae of the brine shrimp Artemia salina. Three proteases, termed A, B and C, have been characterized and partially purified from Artemia salina larvae. Enzyme A is active on benzyloxycarbonyl L-leucin p-nitrophenyl ester (Km = 53 micron, determined at pH 8 and 37 degrees C) but not on alpha-N-benzoyl-DL-arginine p-nitroanilide and is strongly inhibited by micron concentrations of phenylmethylsulfonylfluoride. Enzymes B and C are active on alpha-N-benzoyl-L-arginine p-nitroanilide (Km = 20 and 11 micron, respectively) but not on benzyloxycarbonyl-L-leucine p-nitrophenyl ester and B, but not C, is inhibited by mM concentrations of phenylmethylsulfonyfluoride. Enzymes A, B and C are optimally active at alkaline pH values, do not require either metal ions or -SH groups for their catalytic activity and have molecular weights of 38 000, 33 000 and 34 000, respectively. After heating for 5 min at pH 7.5 and in the presence of 0.7 M KCl half inactivation of proteases A, B and C was attained at 60, 52 and 45 degrees C, respectively.


Purification and properties of an acid protease from human ascitic fluid. An acid protease was isolated from the ascitic fluid of a patient with ovarian cancer. It was purified about 400-fold to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. Its molecular weight was calculated to be 28 000 by gel filtration, and its isoelectric points was found to be pH 4.1. It showed similar activities on acid-denatured bovine serum albumin and on acid-denatured bovine hemoglobin, and its optimal pH for both substrates was 3.0. Sulfhydryl compounds and metal ions had no apparent effects on this enzyme, but pepstatin was strongly inhibitory.


Rate constants of individual steps in papain-catalysed reactions. Rate constants for acylation of papain (EC 3.4.22.2) by specific substrates and its subsequent deacylation are derived from kinetic analysis of the reactions in the presence of aminoacetonitrile and methanol. Methyl and ethyl hippurate and methyl N-benzyloxycarbonylglycinate have marginally higher values of rate constants for acylation than for deacylation, while the reverse is true for ethyl N-benzoyl-L-arginate. Both acylation and deacylation are rate-determining for these substrates, while only deacylation irate-determining for methyl-N-acetyl-L-phenylalanylglycinate. Deacylation is the only rate-determining step for p-nitrophenyl esters of hippuric acid, N-benzyloxycarbonylglycine and N-acetyl-L-phenylalanylglycine. These results are discussed in relation to those from inactivation of the enzyme by alkylating agent in the presence of substrate.


N-Acyl-L-aromatic amino acid deacylase in animal tissues. An enzyme deacylating preferentially N-acyl-L-aromatic amino acids was partially purified from rat kidney. The purification procedure included DEAE-cellulose column chromatography, (NH4)2SO4 fractionation, gel-filtration on a Sephadex G-200 column and further DEAE-cellulose chromatography. The enzyme was thus separated from aminoacylase (N-acylamino-acid amidohydrolase, EC 3.5.1.14) (acylase I). Although the enzyme preparation contained other acylases, the experimental data (effect of p-chloromercuric benzoate, heat stability and inhibition between substrates) suggest that the enzyme acts preferentially on N-acyl derivatives of L-tryptophan, L-tyrosine, L-phenylalanine and L-histidine. This enzyme appears to be present in many animal tissues.


Estimation of Michaelis constant and maximum velocity from the direct linear plot. When estimates of Michaelis-Menten parameters are obtained from kinetic observations taken in pairs, as in the direct linear plot, bias can arise in the final estimates if any pairs lead to negative values of the maximum velocity V. This bias can be removed by treating such negative values as if they were large and positive, and by treating the corresponding values of Km in the same way.


Glutathione peroxidase activities from rat liver. There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.


FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme from Mycobacterium sp. strain Takeo. Purification and some properties. FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme, was homogeneously purified from the particulate fraction of Mycobacterium sp. strain Takeo. The isolated enzyme contains no FAD and few phospholipid, and has a specific activity of 300-360 units/mg of protein. In the assay system without addition of phospholipid (cardiolipin), the enzyme activity was only about 3% of maximum activity. The molecular weight was estimated to be 51 000-55 000 by four methods. Titration by p-chloromercuribenzoate revealed the presence of one cysteine residue/mol of enzyme. The isoelectric point was found to be pH 6.9 by isoelectric focusing. From circular dichroism spectral data, the enzyme protein was found to contain alpha-helix structure of 24%.


The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver. Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.


Properties of taurine: alpha-ketoglutarate aminotransferase of Achromobacter superficialis. Inactivation and reactivation of enzyme. The activity of taurine: alpha-ketoglutarate aminotransferase (taurine: 2-oxoglutarate aminotransferase, EC 2.6.1.55) from Achromobacter superficialis is significantly diminished by treatment of the enzyme with (NH4)2SO4 in the course of purification, and recovered by incubation with pyridoxal phosphate at high temperatures such as 60 degrees C. The inactive form of enzyme absorbing at 280 and 345 nm contains 3 mol of pyridoxal phosphate per mol. The activated enzyme contains additional 1 mol of pyridoxal phosphate with a maximum at 430 nm. This peak is shifted to about 400 nm as a shoulder by dialysis of the enzyme, but the activity is not influenced. The inactive form is regarded as a partially resolved form, i.e. a semiapoenzyme. The enzyme catalyzes transamination of various omega-amino aicds with alpha-ketoglutarate, which is the exclusive amino acceptor. Hypotaurine, DL-beta-aminoisobutyrate, beta-alanine and taurine are the preferred amino donors. The apparent Michaelis constants are as follows; taurine 12 mM, hypotaurine 16 mM, DL-beta-aminoisobutyrate 11 mM, beta-alanine 17 mM, alpha ketoglutarate 11 mM and pyridoxal phosphate 5 micron.


Transient and long-term differential modulations of branched-chain alpha-keto acid decarboxylase activity in hypophysectomized rats. Hypophysectomy caused a marked but transient increase in branched-chain alpha-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain alpha-keto acids. The activities then fall to a new steady state in three weeks with alpha-ketoisovalerate decarboxylase activity within the normal range, alpha-keto-beta-methylvalerate decarboxylase activity at twice normal, and alpha-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.


A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors. Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.


Metabolism and transport of gamma-carboxyglutamic acid. gamma-Carboxyglutamic acid residues have beeh shown to be present in prothrombin, the other vitamin K-dependent clotting factors, and more recently in bone and kidney proteins. This amino acid is formed by a posttranslational vitamin K-dependent carboxylation of glutamyl residues in polypeptide precursors of these protens. It has now been demonstrated that this amino acid, either in the free or peptide-bound form, is not metabolically degraded by the rat, but is quantitatively excreted in the urine. In nephrectomized rats, the tissue concentration of intravenously administered gamma-carboxyglutamic acid is increased, but there is still no evidence of any oxidative metabolism of this amino acid. These amino acid is transported by kidney slices against a concentration gradient, but does not accumulate in liver, intestinal or brain tissues. Preliminary data suggest that gamma-carboxyglutamic acid may be concentrated by a carrier system different from that utilized by other amino acids.


The degradation of normal and analogue-containing proteins in MRC-5 fibroblasts. Proteins containing the arginine analogue canavanine were degraded much more quickly in MRC-5 fibroblasts than those containing only normal amino acids. The degradation of both classes of protein could be well described by a pair of exponential curves, the first representing an early rapid degradation and the second, a slower phase. There were no general trends in the variation with passage number of the cells' ability to degrade either normal or analogue-containing proteins, as judged by the half-lives of proteins. But there was an increase in the proportion of labelled normal protein falling into the early rapid degradation phase as the cells senesced in culture.


Precocious decrease of chymotrypsinogen by thyroxine in the pancreas of suckling mice. Chymotrypsinogen activity in mouse pancreas gradually decreased from birth, reaching the adult level on day 20 after birth. In suckling mice the enzyme activity was decreased to 1/9 of the control value by injection of thyroxine. The activity was not affected by insulin, and was slightly increased, rather than decreased, by daily injection of hydrocortisone. The effect of thyroxine seemed to be direct, not due to modification of adrenal function.


Inhibition of elastolysis by proteinase inhibitors from chick plasma and aorta. Chick plasma contains inhibitor(s) against trypsin and elastase which also appear to retard the degradation of tropoelastin by arterial tissue extracts. Chick aorta extracts also contain similar inhibitors against elastase and trypsin. Both levels of the plasma inhibitor(s) and inhibitor(s) extracted from thoracic aorta increase during early stages of growth and maturation. There is a three- to four-fold increase in the levels of the inhibitor(s) in chick plasma and aorta between one to four weeks after hatching. Of particular interest are the observations that the presence of the inhibitor(s) retards the conversion of soluble elastin (tropoelastin) to smaller elastin peptides. Subsequently, it is speculated that in addition to other vital roles, such proteinase inhibitors may also act in regulating elastogenesis and elastin fiber formation.


Trifolin: a Rhizobium recognition protein from white clover. A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.


Studies on lectins. XLIII. Isolation and characterization of the lectin from restharrow roots (Ononis hircina Jacq.). From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-beta-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (S20,W=6.2 S), polyacrylamide disc electrophoresis at pH 8.6 or 4.5, gel filtration on thin layers of Sephadex G-200 (Mr = 110 000) and dodecyl sulfate electrophoresis (Mr of subunits 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 microgram/ml. The erythroagglutinating activity is not stimulated by Ca2+, Zn2+, Mg2+, Mn2+, Co2+ or Ni2+ salts; it is inhibited most effectively by N-acetyl-D-galactosamine and a number of D-galactose derivatives. Dissociation constants of several lectin . sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph node lymphocytes.


Keratan sulfate-like glycosaminoglycan in the cerebral cortex of the brain and its variation with age. A keratan sulfate-like substance was found kn the cerebral cortex proteoglycans of rats. This substance tripled during the rapid growth phase from 1 to 3 months of age and then decreased steadily to a negligible quantity in the senescent rat, aged 25 months. In contrast, the uronic acid-containing glycosaminoglycans remained constant during the life span studied. The marked decrease observed in the proportion of keratan sulfate-like moiety to the other glycosaminoglycans in the cerebral cortex of the senescent rat (aged 25 months) compared with the young adult rat (aged 3 months) may explain the reduced extracellular volume observed by others in the senescent brain.


Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase. Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetyl-galactosamine 4-sulfate. Fractions, elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.


Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy. The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in laser light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/2H2O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HERPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 microgram protein/ml. At low granule concentrations (15-30 microgram protein/ml) Ca2+-induced aggregation was detected at a threshold of 2-10 mM calcium.


Cluster analysis of respiratory time series. We have investigated the respiratory control system with the hypothesis that, although many variables such as minute ventilation (VI), tidal volume (VT), breathing period (TT), inspiratory duration (TI), and expiratory duration (TE) may be observed, the controller functions more simply by manipulating only 2 or 3 of these. Thus, if tidal volume is the only independent variable, TI being determined by the "off-switch" threshold, these variables should have very similar time courses. Anesthetized dogs were subjected to CO2 breathing and carotid sinus perfusion to stimulate both chemoreceptors. The time series of the variables VI, VT, TT, TE, and TI as well as PACO2 were determined on a breath by breath basis. Derived characteristics of these time series were compared using Cluster Analysis and the latent dimensionality of respiratory control determined by Factor Analysis. The characteristics of the time series clustered into 4 groups: magnitude (of the response), speed, variability and relative change. One respiratory factor accounted for 86% of the variance for the variability characteristics, 2 factors for magnitude (91%) and relative change (85%) and 3 factors for speed (89%). The respiratory variables were analysed for each of the 4 groups of characteristics with the following results: VT and TI clustered together only for the magnitude and relative change characteristics where as TT and TE clustered closely for all four characteristics. One latent factor was associated with the [TT-TE] group and the other usually with PACO2.


Loudness and the power series transformation. Preliminary model and measurements. A nonlinear-algebraic approach to monaural intensity processing is proposed: the purpose is to integrate the conscious loudness attribute evoked by a pure tone with the power-series description of auditory distortion. The preliminary model consists of six postulates leading to a mathematical definition for loudness. To evaluate this equation, two task-specific sets of loudness judgments from each of seven subjects are examined. When linked to loudness-interval responses via the equisection assumption, the equation describes the behaviors quite well. Extrapolations into other intensity ranges predict similar responses with relatively slight overestimates. By relaxing the assumption that subjects will adjust the loudness proportions exactly as instructed, their ratio productions also can be described and predicted with sometimes surprising accuracy. Particularly striking is the prediction of overall levels and the curvilinearities of "doublings" from the "halvings". In addition, the theory proposes absolute loudness measurement, an explanation for the growth of loudness including the principle underlying Steven's Power Law, and might prove useful in examining some exceptions to this relationship. Several aspects of this model differ from traditional approaches to intensity processing, but it appears to warrent further critical evaluations.


Analysis of a cooperative stereo algorithm. Marr and Poggio (1976) recently described a cooperative algorithm that solves the correspondence problem for stereopsis. This article uses a probabilistic technique to analyze the convergence of that algorithm, and derives the conditions governing the stability of the solution state. The actual results of applying the algorithm to random-dot stereograms are compared with the probabilistic analysis. A satisfactory mathematical analysis of the asymptotic behaviour of the algorithm is possible for a suitable choice of the parameter values and loading rules, and again the actual performance of the algorithm under these conditions is compared with the theoretical predictions. Finally, some problems raised by the analysis of this type of "cooperative" algorithm are briefly discussed.


Are there two logistic regressions for retrospective studies? A comparison is made between two different approaches to the linear logistic regression analysis of retrospective study data: the prospective model wherein the dependent variable is a dichotomous indicator of case/control status; and the retrospective model wherein the dependent variable is a binary or polychotomous classification of exposure. The two models yield increasingly similar estimates of the relative risk with increasing degrees of covariate adjustment. When the covariate effects are saturated with parameters, the relative risk estimates are identical. The prospective model is generally to be preferred for studies involving multiple quantitative risk factors.


A graphical aid for determining sample size when comparing two independent proportions. Standard sample size calculations for n, the number of observations per group when comparing two independent proportions, P1 and P2, require the specification of four quantities: P1, one of the two proportions of interest; delta = P2 - P1, the smallest difference which it is important to detect; alpha, the significance level; and beta, the chance of failing to detect a difference as large as delta. In terms of these four quantities, the graphical aid is a series of charts showing isographs of sample size for selected values of n ranging from 35 to 500. The isographs, i.e. curves connecting points of equal sample size, are based on the asymptotic arc sine approximation and are plotted on the grid formed by P1 on the abscissa and delta on the ordinate. Eight separate charts are available for different choices of alpha and beta. These charts are especially useful in situations where the feasible sample size is roughly known, in which case the detectable difference, delta, can be read directly from the graph.


A genetic model involving fetal effects on traits of the dam. The genetic fetal effects model shows that the usual sire effect is composed of one-half the direct additive genetic value and one-fourth of the fetal additive genetic value of the sire. The usual sire component of variance is actually the variance of that function. Genetic covariances between records of relatives influenced by fetuses of related sires can easily be written. If the magnitude of fetal sire effects is such that nonrandom use of fetal sires on daughters of sires being evaluated on daughter performance results in bias, the bias can be eliminated (Henderson 1975) by considering the fetal sire effects to be fixed effects. Some reduction in prediction error variance is likely by including fetal sire in the sire evaluation model.


Regression analysis of grouped survival data with application to breast cancer data. Use of the proportional hazards regression model (Cox 1972) substantially liberalized the analysis of censored survival data with covariates. Available procedures for estimation of the relative risk parameter, however, do not adequately handle grouped survival data, or large data sets with many tied failure times. The grouped data version of the proportional hazards model is proposed here for such estimation. Asymptotic likelihood results are given, both for the estimation of the regression coefficient and the survivor function. Some special results are given for testing the hypothesis of a zero regression coefficient which leads, for example, to a generalization of the log-rank test for the comparison of several survival curves. Application to breast cancer data, from the National Cancer Institute-sponsored End Results Group, indicates that previously noted race differences in breast cancer survival times are explained to a large extent by differences in disease extent and other demographic characteristics at diagnosis.


The estimation of false negatives in medical screening. In a medical screening program for early detection of disease, one or more screening modes are administered to an apparently healthy population. Knowledge of the true disease status for all screened individuals would allow estimation of the false negative and false positive rates for each mode of detection and for the program as a whole. This paper develops capture-recapture methods applicable to programs as a whole. This paper develops capture-recapture methods applicable to programs when follow-up of individuals negative on screening is not performed or is incomplete. The methods require at least two independent modes of detection. Data from a breast cancer screening program illustrate the procedure. The results of four screening examinations at approximately one-year intervals and the long-term follow-up of all screened individuals support the usefulness of these methods in the evaluation of a screening program.


Role of voltage-sensitive receptors in nicotinic transmission. This paper compares the conductance induced by bath-applied acetyl-choline (ACh) and by the same transmitter released from nerve terminals at Electrophorus electroplaques. For the former case, dose-response relations are characterized by the maximal agonist-induced conductance, rgamma (130 mmho/cm2), and by the concentration which induces half this conductance; this concentration is termed Kapp and equals 50 micron at -85 mV. For the latter case, neurally evoked postsynaptic currents (PSCs) are characterized by the peak conductance during strongly facilitated release, gPSC, and by the rate constant for decay, alpha. Since gPSC roughly equals rgamma, it is concluded that the PSC activates nearly all available receptor channels. These and other data agree with recent estimates that during the growth phase of the quantal response, (a) the ACh concentration is at least several hundred micromolar; and (b) most nearby channels are activated. However both alpha and Kapp increase during depolarization, at a rate of about e-fold per 86 mV. These observations on voltage sensitivity suggest that a suprathreshold synaptic event is rapidly terminated because the action potential abruptly releases ACh molecules from receptors.