Abnormal induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in leukocytes from subjects with heterozygous familial hypercholesterolemia. Human leukocytes isolated from fresh defibinated blood were shown to utilize acetate and mevalonate for sterol synthesis. The capacity of the leukocytes to synthesize sterols is limited severely as compared to their ability to convert mevalonate into farnesyl pyrophosphate (which they hydrolyze rapidly to free farnesol) and into squalene. When leukocytes are incubated in a medium containing lipid-free serum, synthesis of sterols from acetate, but not from mevalonate, is much enhanced. It was shown that this increased synthesis resulted from increased levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in the cells. A comparison was made of the activation of sterol synthesis from acetate in leukocytes of normal individuals and of heterozygous familial hypercholesterolemics. The latter group responded to incubation in lipid-free sera with a significantly higher activation than the cells of normocholesterolemics. This activation was shown to be well correlated with a higher induction of 3-hydroxy-3-methylglutaryl-CoA reductase in the heterozygous cells than in the normals. The leukocytes of a heterozygous familial hypercholesterolemic individual were found to release, into a lipid-free incubation medium, more endogenously synthesized [3H]sterol (but not [3H]squalene) than the cells of a normal person. It is suggested that the genetic abnormality in heterozygous familial hypercholesterolemia could be accounted for by a mutation resulting in a weaker binding of a sterol repressor by heterozygous cells than by normal cells.


The use of 13C nuclear magnetic resonance of aromatic amino acid residues to determine the midpoint oxidation-reduction potential of each iron-sulfur cluster of Clostridium acidi-urici and Clostridium pasteurianum ferredoxins. 13C NMR of the aromatic residues of Clostridium acidi-urici [Phe2]ferredoxin (a chemically modified ferredoxin in which a phenylalanyl residue replaces a tyrosyl residue) and Clostridium pasteurianum ferredoxin permits one to distinguish and probe each iron-sulfur (Fe7S7) cluster neighboring the aromatic residues within each protein. This is because the ring carbon resonance shifts of the phenylalanyl and tyrosyl residues can be distinguished. The 13C NMR results suggest that the midpoint oxidation-reduction potentials of the two Fe4S4 clusters in C. pasteurianum and C. acidi-urici ferredoxin differ by 10 plus or minus 5 mv and smaller than mv, respectively. 13C NMR of an equilibrium mixture of methyl viologen-reduced C. acidi-urici and C. pasteurianum ferredoxin shows that the protein midpoint oxidation-reduction potential of C. acidi-urici ferredoxin is 47 plus or minus 10 mv lower than that of C. pasteurianum ferredoxin. We attribute the differences in cluster and protein midpoint oxidation-reduction potentials to differences in protein structure.


Heterogeneity of the carbohydrate chains of sulfated bronchial glycoproteins isolated from a patient suffering from cystic fibrosis. Sulfated glycoproteins having blood group H activity were isolated from the sputum of a child suffering from cystic fibrosis, by reduction of the fibrillar mucus, chromatography on ECTEOLA-cellulose, and gel filtration on Sepharose 4B. The sulfated glycoproteins were degraded with alkaline borohydride, and the degradation products were fractionated by chromatography on ion exchange resins and by gel filtration. The carbohydrate chains thus obtained have a wide heterogeneity with regard to acidity and molecular size. The neutral chains contain blood group H active oligosaccharides and incomplete chains as short as 1 residue of 2-acetamido-2-deoxy-D-galactose. The minimal size of the neuraminic acid-containing chains is less than that of the sulfated chains, which increases with the degree of sulfation. The sulfate groups are linked at C-6 at the D-galactose residues.


Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins. At least 60 percent of the fucose residues in HeLa cell glycoprotein are nonreducing, terminal, and closely proximal to the protein carbohydrate linkage. As determined by pulse-labeling with (3H) fucose and sizing glycopeptides in Sephadex chromatography these residues are added near the time of completion of oligosaccharide chains. Glycoproteins, the large bulk if not the only macromolecules labeled with radioactive fucose in HeLa cells, were not soluble in ethanol or chloroform-methanol, 2:1, but were substanially solubilized by chloroform-methanol-water, 10:10:3. Folch extraction of labeled cells and analysis of the upper phase revealed little if any (3H) fucose-labeled glycosphingolipids. Studies on the distribution of radioactively labeled glycoprotein in various cell fractions show that in uniform labeling conditions fucosylated glycoproteins accumulate in the plasma membrane specifically. Pulse-chase and protein synthesis inhibitor studies show that there is an internal pool of completed fucosylated glycoprotein, taking not less than 12 min to deplete. From this pool newly synthesized glycoprotein moves to the plasma membrane with a transit time of 12 min and little was found soluble in the cell. By contrast, a pool of protein labeled with 14C-aminoacids and precursor to plasma membrane protein is small and depleted almost immediately. From this pool newly synthesized protein molecules move to the plasma membrane with a transit time of less than 2 min. It would appear that these two distinct molecular components of plasma membranes may be assembled into membranes sequentially or into the plasma membrane independently.


Prothrombin fragments. Ca2+ binding and activation kinetics. The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).


Multiple forms of cytochrome P-450 in phenobarbital- and 3-methylcholanthrene-treated rats. Separation and spectral properties. Multiple forms of liver microsomal cytochrome P-450 isolated from immature male rats pretreated with phenobarbital or 3-methylcholanthrene are described. Afraction of low specific content (Fraction A. 1.7 TO 4.0 nmol of cytochrome P-450 per mg of protein) and a fraction substantially purified (Fraction B, 9.0 TO 11.0 NMOL of cytochrome P-450 per mg of protein) are obtained by DEAE-cellulose chromatography of a partially purified cytochrome P-450 preparation in the presence of Emulgen 911. Shifts in the absorption maxima in the CO-reduced and ethyl isocyanide difference spectra are observed in the fractions derived from 3-methylcholanthrene-treated rats. The fractions derived from phenobarbital-treated rats exhibit different 455:430 ratios and pH intercepts in the ethyl isocyanide difference spectra. The absolute oxidized spectra and n-octylamine binding spectra at room temperature and EPR analysis at the temperature of liquid helium characterize all the fractions, except the Fraction A from 3-methylcholanthrene-treated rats, as low spin ferric hemeproteins. The A hemeprotein fractions from both 3-methylcholanthrene- and phenobarbital-treated rats have poor catalytic activity for the metabolism of benzphentamine and 3,4-benzo-[a]pyrene in comparison to the B hemeprotein fractions which may be due to the presence of a high concentration of Emulgen 911 in the A fractions. However, the presence of Emulgen 911 cannot account for the spectral differences among the fractions.


Studies on the mechanism of covalent incorporation of a lysergyl derivative to immunoglobulin peptides in vitro. In vitro incubation of hyperimmune rabbit lymphoid cells with the hallucinogenic indole alkaloid, d-lysergic acid diethylamide (LSD), results in biosynthesis of modified, secretable immunoglobulin peptides. Modification involves covalent attachment of the lysergyl moiety to COOH-terminal portions of the peptides. Aalogous effects occur when cells are incubated in vitro in the presence of the non-hallucinogenic LSD analogue, d-lysergic acid, and N-[3H]-carboxymethyl-d-lysergamide. The phenomenon is reversed by tryptophan and is inhibited by puromycin and cycloheximide. In vitro attachment of the lysergyl moiety occurs in the presence of actinomycin D at levels which inhibit RNA synthesis. While LSD is not attached to intracellular tRNA, the drug binds to 80 S ribosomes from hyperimmune lymphoid cells with high affinity (K A equals 3.5 times 10-8 M-1). Similar binding occurs to nonimmune splenic ribosomes. Implications of these findings are discussed with respect to the degree of involvement of cellular protein translational mechanisms in the covalent attachment of the lysergyl moiety to low molecular weight immunoglobulin peptides.


Sequential changes in the protein synthetic activity of male Xenopus laevis liver following induction of egg-yolk proteins by Estradiol-17 beta. Administration of a single injection of estradiol-17 beta to adult male Xenopus laevis induces the synthesis and secretion by the liver of the egg-yolk protein complex vitellogenin. Explants taken from livers of hormone-treated animals and maintained in organ culture continue to produce this protein at a rate which is dependent on the length of time between estrogen injection of the donor and preparation of the explants. Organ culture, therefore, can be used to quantitate the vitellogenic response and to study its time course in detail. An increase in the rate of incorporation of radioactive amino acids into secreted protein is first detectable in explants taken from animals 12 hours after estradiol injection. The response becomes increasingly pronounced as the period after hormone treatment of the donors is prolonged, and it is maximal in explants taken from frogs 12 days after estrogen administration. At this time production of vitellogenin can account for as much as 90 per cent of total protein synthesis in the liver. Labeling of intracellular protein with [14C]serine is stimulated 2-fold and incorporation into secreted protein increased 36-fold under conditions where the rate or extent of uptake of radioactive precursor into the acid-soluble pool does not change. After a lag period of 2 to 4 hours the secretion of vitellogenin in culture continues at a constant rate for up to 3 days. At least part of the stability of yolk protein synthesis in culture is attributable to long lived messenger RNA species. The major component of vitellogenin labeled wither in vivo or in culture has a molecular weight of approximately 180,000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Material labeled with [3H]serine gives a similar distribution of radioactivity on gel electrophoresis as that labeled with [35S]methionine, with no peak corresponding to the molecular weight of phosvitin. It is suggested that phosvitin is not a primary product of translation but is synthesized as part of the large subunit of vitellogenin.


Glucocorticoid inhibition of protein synthesis in vivo and in vitro. In young adult rats receiving glucocorticoids in pharmacological amounts, the accumulation proteins in the liver was suppressed. In vivo pulse-labeling experiments showed that inhibition of protein synthesis in the liver was an early effect of a single dose of glucocorticoid followed by a stimulation of protein synthesis. In animals receiving multiple doses of glucocorticoid over a period of a few days, a more pronounced inhibition in protein synthesis was observed, and the secondary stimulation of protein synthesis did not occur. These in vivo observations were paralleled by the results with cell-free protein-synthesizing systems, which indicated that the glucocorticoid inhibitory effect was imparted to the soluble fraction of the liver.


On the sequence of reactions leading to cytochrome P-450 synthesis-effect of drugs. The effect of phenobarbital on the rates of the synthesis of the protein and heme moieties of cytochromeP-450 has been studied. For this purpose, cytochrome P-450 has been partially purified as its P-420 derivative and the labeled amino acid incorporation into the protein has been studied after subjecting a partially purified preparation to sodium dodecyl sulfate gel electrophoresis. The incorporation studies into the protein species after sodium dodecyl sulfate gel electrophoresis reveal that the drug primarily accelerates the rate of apoptotein synthesis followed by an increase in the rate of heme synthesis. The messenger for apocytochrome P-450 appears to be fairly stable.


Studies on a 15-hydroxyprostaglandin dehydrogenase from human placenta. Purification and partial characterization. A 15-dyroxyprostaglandin dehydrogenase has been purified from human placenta to apparent monodispersity. The reaction catalyzed by this enzyme is freely reversible with an equilibrium constant of approximately 6.5 times 10-8 M. The activation energy is 9900 calories per mol. The molecular weight of the enzyme determined by gel filtration is 51,500; sodium dodecyl sulfate disc gel electrophoresis gives a value of 42,000. No evidence was obtained for the existence of multiple forms of the enzyme or for subunits.


The degradative effects of porphyrins and heme compounds on components of the microsomal mixed function oxidase system. The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:1 or 1:1), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoprophyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b5 and 14C-labeled heme, Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b5, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphvrin IX and mesoporphyrin IS could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in the presence of deuterium oxide. It is suggested that the mechanism by which porphyrins inhibit the mixed function oxidase system is through porphyrin-sensitized photo-oxidation of various constituents of the hepatic microsomal fraction and that the formation of singlet oxygen molecules is most likely involved in this process. Moreover the destructive effects of heme compounds on the microsomal components and activities of the drug-metabolizing mixed function oxidase system raise questions concerning the hypothesis that the components of this system, and specifically cytochrome P-450, are involved in the activity of the heme oxygenase system.


The control of the synthesis of pyruvate carboxylase in Pseudomonas citronellolis. Evience from double labeling studies. The level of pyruvate carboxylase in Pseudmonas citronellolis is controlled by the carbon source of the growth medium. The activity of the enzyme is highest in cells grown on lactate or glucose and virtually absent in cells grown on malate or aspartate. Double labeling studies with 3H- and 14C-labeled leucine confirm that pyruvate carboxylase is synthesized in the presence of lactate but not in the presence of aspartate. The studies also show that coordinated regulation occurs at the level of the synthesis of the two polypeptides which make up pyruvate carboxylase in P. citronellolis, rather than at the stages of their assembly into protomers or the biotinylation of the apoenzyme. There is no evidence for control of the catalytic acitivity of the holoenzyme via effectors. In all other varieties of pyruvate carboxylase examined thus far, the enzyme appears to be constitutive with regulation accomplished either through effector modulation of holoenzyme activity (pyruvate carbox-lase from animal sources, yeast, several species of bacteria) or through control of the biotinylation of the apoenzyme by holocarboxylase synthetase (Bacillus stearothermophilus, yeast).


A theoretical model for the covalent assembly of immunoglobulins. Application to the assembly of human immunoglobulin G in vitro. A simple theoretical model for the formation of interchain disulfide bonds (covalent assembly) in immunoglobulins has been developed. This model successfully simulates the experimentally determined sequence of disulfide bond formation in vitro for human immunoglobulin G1 and G4 (Petersen, J. G. L., and Dorrington, K. J. (1974) J. Biol. Chem. 249, 5633-5641). The model appears to be generally applicable suggesting that the various pathways of covalent assembly observed in vivo and in vitro reflect structural differences between the immunoglobulin classes and subclasses. The rate of assembly, however, is dependent upon environmental factors.


A new look at osteogenesis imperfecta. A clinical, radiological and biochemical study of forty-two patients. In a clinical, radiological and biochemical study of forty-two patients from Oxford with osteogenesis imperfecta, it was found that patients could be divided simply into mild, moderate and severe groups according to deformity of long bones. In the severe group (seventeen patients) a family history of affected members was uncommon and fractures began earlier and were more frequent than in the mild group (twenty-two patients); sixteen patients in the severe group had scoliosis and eleven had white sclerae; no patients in the mild group had white sclerae or scoliosis. Radiological examination of the femur showed only minor modelling defects in patients in the mild group, whereas in the severe group five distinct appearances of bone (thin, thick, cystic and buttressed bones, and those with hyperplastic callus) were seen. The polymeric (structural) collagen from skin was unstable to depolymerisation in patients in the severe group, but normal in amount, whereas the reverse was found in the mild group. This division according to long bone deformity may provide, a basis for future research more useful than previous classifications.


Equinus deformity due to haemangioma of calf muscle. Joint deformity secondary to extensive haemangiomatous involvement of the soft tissues has been well described and is easy to diagnose. If the haemangioma is small, localised and within the belly of a muscle the diagnosis is more difficult. In equinus deformity of obscure aetiology localised calf tenderness may be the only diagnostic sign. Three children with equinus deformity caused by a small haemangioma in the calf muscles were treated by simple excision with satisfactory results.


Reflections upon the aetiology of congenital pseudarthrosis of the clavicle. With a note on cranio-cleido dysostosis. The cause of pseudarthrosis of the clavicle is obscure. Right-sidedness is an almost constant feature. We have proposed that the lesion is sometimes due to pressure upon the developing clavicle by the subclavian artery which is normally at a higher level on the right side. This may be accentuated in the presence of cervical ribs or unduly elevated first ribs, both of which we have observed in association with pseudarthrosis. We have also noted pseudarthrosis on the left side in association with dextrocardia (when the relative positions of the subclavian arteries are reversed) and in the presence of a large left cervical rib. We have speculated upon the nature of the clavicular defect in cranio-cleido dysostosis, in which disorder the first ribs are habitually elevated. A similar mechanism may be involved.


The management of local complications of total hip replacement by the McKee-Farrar technique. One thousand and forty-two McKee-Farrar prostheses of the present design inserted in Norwich from January 1965 to December 1972 have been reviewed retrospectively to determine the incidence of complications needing revision. Of prostheses implanted for more than two years, 6-6 per cent needed revision for loosening (cup 3-5 per cent; stem 2-2 per cent; both components 0-9 per cent). Of the total number, 2-3 per cent became infected and 1-9 per cent dislocated. Most dislocations needed only a single closed reduction but 0-8 per cent were revised. The outcome of revision operations was also assessed. Of revisions for loosening, 40 per cent needed no further operation but 23 per cent required excision; pelvic fracture or bone destruction around the components made success unlikely. Revisions for dislocation were disappointing. Of all revisions 17 per cent became infected. Excision arthroplasty is better than a series of failed revisions in an elderly patient.


Acetabular development in congenital dislocation of the hip. With special reference to the indications for acetabuloplasty and pelvic or femoral realignment osteotomy. This investigation examined the validity of the hypothesis that the acetabulum in congenital dislocation of the hip will develop satisfactorily provided accurate congruous and concentric reduction is obtained as early as possible, and is maintained throughout growth. Seventy-two patients with eighty-five hips were studied. The children were more than one year old on admission and over ten years at the time of review. Acetabular development was assessed radiologically by measurement of the acetabular angle. Angles of less than 21 degrees were regarded as normal, and more than 21 degrees as indicating some failure of development. Satisfactory acetabular development occurred in 80 per cent (angles 24 degrees or below), and was unsatisfactory in 20 per cent (angles above 24 degrees). If three errors in management, namely failure to obtain congruity, failure to maintain congruity and ischaemic necrosis secondary to manipulative reductions, are excluded from the analysis, it is found that 95 per cent of acetabula develop satisfactorily. The outcome is largely independent of the age on admission up to four years old, and of bilateral involvement. It is concluded that acetabuloplasty should not be necessary if the patient is admitted under the age of four or congruity is obtained in the functional position under four and a half years.


The Walldius hinge arthroplasty.

The Walldius hinge arthroplasty. Eighty-three Walldius arthroplasties, performed by one surgeon as salvage operation on the knee joint between 1966 and 1972, were independently reviewed. The fifty-seven living patients with sixty-seven arthroplasties were interviewed and examined and the clinical records of the deceased patients were inspected. Sixty-seven arthroplasties (81 per cent) were successful and sixteen failed (19 per cent). Acrylic cement was used to secure the prosthesis on eight occasions only. There were two primary infections (2-4 per cent) and two delayed (2-4 per cent). Major loosening occurred in three arthroplasties (3-6 per cent). Minor loosening was compatible with a good result. Arthrodesis was successful on the two occasions on which it became necessary to remove the implant. There were no disasters. On the basis of these results it is considered that the Walldius arthroplasty can justifiably be offered as an alternative to primary arthrodesis of the knee.


Popliteal cysts in children. The case against surgery. The natural history of 120 popliteal cysts in children has been reviewed. Of seventy untreated cysts fifty-one disappeared spontaneously during a mean period of one year and eight months. Of fifty cysts submitted to operation, twenty-one recurred in a mean period of seven months. Three children with recurrences had more than one further operation. Most popliteal cysts in children disappear spontaneously, and operation without very good reason is unjustified.


Link arthroplasty of the metacarpo-phalangeal joints. A preliminary report of a new method. Link arthroplasty is a system of joint replacement in which the joint is left almost intact with no great removal of bone. It is based on a two-piece self-locking hinge slotted into the metacarpal head and phalangeal shaft. The operation is simple and no special instruments are needed. The preliminary follow-up of fifty-four metacarpo-phalangeal joint replacements showed thirty-five good and sixteen fair results.


Benign chondroblastoma of bone. Report of a case. A benign chondroblastoma of bone is reported. It was unusual because it occurred in an old lady, in a toe, and it was not painful and radiologically resembled a chondroma. The coarsely lobulated tumour showed a varied microscopic appearance, but it consisted chiefly of closely packed sheets of small, round polygonal or fusiform cells. There was some calcification present. The literature is reviewed.


Hangman's fracture.

Hangman's fracture. Of four hangman's fractures of the axis, three occurred in road accidents and were stable, undisplaced and free of neurological signs, with full recovery after six to twelve weeks in a cervical collar. The fourth fracture occurred in a fall with profound tetraparesis from haemorrhage into the spinal cord, and the patient died a week later. There are two types of hangman's fracture: that of the axis pedicle, which results from extension and compression of the cervical column, is more common than usually supposed because of the slight symptoms it causes.


An investigation of the centres of pressure under the foot while walking. The forces under the foot while walking have been measured using a high sensitivity force-plate of the strip-suspended type combined with simultaneous filming of the sole of the foot. The recording of data and the calculation and plotting of results were much simplified by computer aid. Normal and abnormal feet, both barefoot and shod, were investigated in sixteen subjects. It was found that in normal barefoot walking the forefoot carried a total load of the order of three times that of the heel. When footwear was worn the function of the forefoot was progressively reduced as the rigidity of the sole of the shoe increased. Painful conditions of the forefoot also produced a large reduction in the proportion of the total load transferred.


Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.


Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins. Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient  oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.


Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver. Lipoprotein particles (d less than 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800  A in diameter (mean similar to 420 A); the smooth er particles 200-900 A (mean similar to 520 A); the Golgi particles 350-950 A (mean similar to 580A); and serum VLDL 300-800 A (mean similar to 450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex. At short intervals (less than 15 min), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover. When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum. It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles-presumed to be precursors of liver VLDL-begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.


Evidence for firm linkages between microtubules and membrane-bounded vesicles. Direct evidence is presented in support of the widely held idea that membrane-bounded vesicles can bind firmly to microtubules. This is shown in P. caudatum which contains ribbons of straight microtubules located in open cytoplasm and uniquely associated with the disk-shaped vesicles. These vesicles frequently lie flat against the face of the ribbons at a constant distance of 30-40 nm. Under certain conditions the ribbons are compressed into zigzag pattern, but the vesicles continue to maintain their 30-40 nm spacing with the tubules and The author's interpretation of this phenomena is that the vesicles and the microtubules are strongly bound together. This interaction appears to be via a filamentous material rather than bridges.


Gas-liquid chromatographic studies of reactions and structural relationships of steroids. II. Positions 3, 11, and 20 in the pregnane series. Qualitative and quantitative effects of classical reactions on steroids observed by gas-liquid chromatography (GLC) under standardized conditions, including the double internal standard technique, are reported. Simple procedures applicable to nanogram amounts of reactants which afford excellent yields of the major products are described. Reactions studied include the Wolff-Kishner removal of keto groups (WK), their conversion to hydroxyl groups by NaBH4(RD), and to dioxolone derivatives by ethylene glycol (DO); the conversion of hydroxyl to keto groups by CrO3 (OX), and to TMS ethers by hexamethyldisilazane; the hydrolysis of dioxolone and TMS derivatives by H+ (HY). GLC chromatograms of reaction mixtures of single and multistep reactions readily provide information on effects on functional groups at positions 3, 11, and 20 in the pregnane series, and the retention times of many steroids unavailable from commercial or other sources. GLC data analysis provides relationships between steroid structure and retention time from which methods for the computation of retention times and for steroid identification are designed. The accuracy of the computation methods is demonstrated.


Determination of carbamazepine and its epoxide metabolite in plasma by high-speed liquid chromatography. A liquid-chromatographic method for the simultaneous determination of carbamazepine and its active metabolite (carbamazepine 10,11-epoxide) in plasma has been developed. The two compounds were identified in plasma by mass spectrometry. The lower limit of sensitivity is about 4 and 40 ng for the drug and its metabolite, respectively. 10,11-Dihydrocarbamazepine is used as internal standard for the determinations, which have precisions of 2.2 and 4.2%, respectively. No derivatization is needed. The specificity of the method for carbamazepine is shown by the significant correlation (r = 0.99) between the results obtained by this method and by mass fragmentography for the drug in plasma of patients.


Automated single-column analysis of amino acids using ascorbic acid as reductant for air-stable ninhydrin. The procedure for operation of a constant-temperature, single-column automated amino acid analyser in the sub-nanomole range is described. The cycle time for a complete analysis is 90 min including equilibration for next cycle. Eluting buffers can be made in the laboratory or commercially available concentrates (Pico-Buffers) can be used. A novel reducing agent, ascorbic acid, incorporated into the column buffers was used to reduce air-stable ninhydrin.


Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. III. Rigid agarose gels cross-linked with divinyl sulphone (dvs). Agarose cross-linked with divinyl sulphone (DVS) is a new matrix for chromatography and immobilized enzymes that has distinct advantages over common agarose gels. It has outstanding mechanical stability as compared with these gels, and the rigid gel beads form beds permitting very high flow-rates. In addition, DVS-agarose is superior to agarose gels with respect to chemical stability in acid and neutral media. In alkaline solutions above pH 8, there is a slow elimination of the sulphone-containing bridges, but without noticeable concomitant dissolution of the gels below pH 12 for moderately or highly cross-linked gels. The DVS-agarose is sufficiently thermostable to be heated in an autoclave.


Specific quantitative gas-liquid chromatographic analysis of methyldopa and some foreign related amino acids in raw material and commercial tablets. A relatively simple gas-liquid chromatographic (GLC) procedure has been designed for the rapid detection and accurate quantitation of some theoretically possible foreign related amino acid contaminants in alpha-methyldopa raw material and commercial tablets. After trimethylsilylation of the drug or drug mixture with N,O-bis(trimethylsilyl)acetamide in acetonitrile at ambient temperature for 90 min, the derivatives are eluted from a methylsilicone column isothermally at 170 degrees. Quantitation of the components is effected by simple computation relative to dibenzyl succinate as the internal standard. The results obtained by applying the GLC procedure to the analysis of a number of multicomponent synthetic mixtures are in good agreement with the theoretical values. The percentage of label claim values obtained by the GLC method for commercial tablets are compared to those measured colorimetrically by the official U.S.P. procedure. No foreign related amino acid impurities were detected in any of the three commercial dosage forms examined.


Ionic effects in the chromatography of proteins on columns of controlled-pore glass. Bovine serum albumin, acetylcholinesterase and two non-protein markers were chromatographed on columns of controlled-pore glasses in buffer solutions of varied ionic strengths. Adsorption of protein to the glass and ionic effects due to repulsion of the negatively-charged solutes from the glass matrix were observed. When glasses were treated with Carbowax 20M, the adsorption disappeared but ion exclusion effects persisted. The ionic behavior of Carbowax-treated glasses was similar to that observed by one of the authors when using agarose columns.


Thin-layer chromatographic separation of methadone and its primary metabolite in the presence of other drugs in urine specimens. Three solvent systems for thin-layer chromatography have been developed for methadone and its primary metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine). These solvent systems separate methadone and its primary metabolite, and are not interfered by other drugs of abuse in urine specimens.


The removal of organic matter with a weakly basic macroreticular ion-exchange resin. A case history. Natural waters containing organic pollutants have a strong tendency to foul anionic exchange resins irremediably. The use of various types of scavenging resins is discussed and results are given on trials carried out at a chemical plant. The conclusion is drawn that a proper analysis of organic matter present in water cannot be made and that consequently it is difficult to determine which types of scavengers are the most suitable. The paper suggests a principle of calculating weakly basic macroreticular resins into the flow system so as to function both as a scavenger and a weakly basic ion-exchange resin.


Sterols of the cockle Cerastoderma edule. Evaluation of thermostable liquid phases for the gas-liquid chromatographic-mass spectrometric analysis of the trimethylsilyl ethers of marine sterols. Some recent liquid phases for gas-liquid chromatography with improved thermostability have been evaluated for their use in the gas-liquid chromatographic-mass spectrometric (GC-MS) analysis of the complex mixtures of sterol trimethylsilyl ethers obtained, often in small amounts, from marine organisms. A combination of Dexsil 300 GC, SE-30 "ultraphase" and Silar 5CP is recommended for use in this technique because of the excellent separating efficiency and the very low substrate bleed. GC-MS analysis of the sterol trimethylsilyl ethers obtained from the cockle Cerastoderma edule has established the identity and relative proportions of the eleven sterols present.


Analysis of the diazinon metabolites G 27550 and GS 31144 by gas-liquid chromatography with nitrogen-specific detection after derivatization. The alkylation and silylation of the diazinon metabolites G 27550 (2-isopropyl-4-methyl-6-hydroxypyrimidine) and GS 31144 [2-(1'-hydroxy-1'-methyl)-ethyl-4-methyl-6-hydroxypyrimidine] with methyl iodide/sodium hydride and bis-(trimethylsilyl)-trifluoroacetamide, respectively, were examined for use as confirmation techniques for these metabolites in sample material. The methods proved to be rapid and simple. The derivatives chromatographed well on 4% SE-30/6% QF-1. Electrolytic conductivity detection proved satisfactory for the derivatives with minimum cleanup. The techniques were successfully applied to the analysis of the two metabolites in dog urine in the range of 1 ppm.


Mass spectrometric identification of the thermal decomposition products of N-(p-toluenesulfonyl)-N'-(aza-bicyclo-(3.3.0.) octyl) urea (S-852). A study was made on the thermal decomposition products of S-852. The identification of the degradation products separated on a 3% OV-17 glass column was obtained by means of the mass spectrometric technique


Changes in plasma growth hormone levels in normal and acromegalic subjects following administration of 2-bromo-alpha-ergocryptine. 2-Bromo-alpha-ergocryptine (CB-154), AN ERGOT ALKALOID THAT STIMulates dopaminergic receptors, caused a widely varying, but significant increase in plasma GH levels in normal subject whereas a marked and protractedfall in values was observed in some patients with acromegaly. It is suggested that these effects were mediated by activation of either hypothalamic or hypophyseal dopaminergic receptors. The fact that GH values fell to or near normal levels in 4/8 cases suggests that CB-154 may have therapeutic possibilities in acromegaly.


Growth hormone responses following double pulse oral glucose administration in various clinical states. The 8-h double pulse oral glucose test is proposed as an alternative screening procedure for determining the adequacy of growth hormone (GH) release. The second pulse of glucose is timely in suppressing GH release and delaying it for a more predictable controlled elevation. Peak GH (mean plus or minus SD) values following the double pulse glucose test were 17 plus or minus 10.2, 16.5 plus or minus 2.2, and 1.3 plus or minus 0.5 ng/ml in normal controls, short stature patients and GH deficient patients, respectively. Peak GH values following insulin hypoglycemia were 36.9 plus or minus 13.8, 21.9 plus or minus 23.0 and 1.8 plus or minus 1.1 ng/ml in normal controls, short stature patients and GH-deficient patients. Peak GH values during a 5-h oral glucose tolerance test were 16.4 plus or minus 6.0 and 10.2 plus or minus 3.3 ng/ml in normal controls and short patients, respectively. These differences in peak GH values between the various clinical and control groups were not significantly different except for the greater GH peak values reached in short patients in the double pulse glucose test compared to the 5-h oral glucose tolerance test. The rise in GH following double pulse oral glucose is more timely predictab-e than after the 5-h oral glucose test and involves less professional time than the insulin tolerance test as it does not require close medical supervision.


The excretion of 2-hydroxyestrone during the menstrual cycle. A double isotope derivative determination technique was developed to investigate the excretion of 2-hydroxyestrone inhuman menstrual cycle. The method is highly specific, accurate and precise within the range of 0.2-20 nmol 2-hydroxyestrone/20ml of urine. The amounts of 2-hydroxyestrone excreted during menstrual cycle varied between 10 (proliferation phase) and 60 mug/24 h urine (ovulatory peak), which is comparable to that of estriol, supposed hitherto to be the main excretion product of estrogen metabolism.


Aromatization of androstenedione by isolated human hairs. The formation of (3-H) estrone has been demonstrated in human anagen scalp hair roots incubated with (1, 2, 6, 7-3H) androstenedione, and approximate rates of formation of 0.2 pmol of estrone/mg DNA/h were observed. Thus, hair is a potential site for the extraglandular formation of estrogen in man.


Effect of alanine and glycine on glucagon secretion in postabsorptive and fasting obese man. Changes in blood levels of glucagon, insulin and glucose in response to infusions of alanine and glycine have been studied in postabsorptive and fasting obese human subjects. Four-to-five-fold elevations of baseline plasma alanine levels stimulated glucagon secretion significantly. Supraphysiological plasma levels of glycine had a small but insignificant stimulatory effect on glucagon secretion. The glucose increase (6 to 10 mg per 100 ml) observed within 30 min of a supraphysiologic alanine infusion in subjects fasted for 2 or more weeks may be secondary to glucagonmediated glycogen breakdown. However, despite equivalent glucagon rises in the other two study periods, no significant rise in blood sugar was noted during the period of infusion.


Intra-uterine treatment of a hypothyroid fetus. A woman in her 24th week of gestation was referred for treatment of hypothyroidism, after she underwent radioablation of the thyroid during the 13th week of gestation. Because of the high risk of hypothyroidism in the fetus, prenatal administration of intramuscular T-4 to the fetus was begun at 32 weeks. The last dose of T-4 was given 2 weeks before delivery; cord blood levels of T-4 and T-3 were undetectable and the TSH concentration was markedly elevated. The case illustrates several important physiological concepts regarding thyroid hormone and TSH metabolism in the fetal-placental unit, including the minimal placental permeability to iodothyronines and TSH, independent function (including feedback control) of the fetal hypothalamic-pituitary-thyroid axis, and the TSH response at parturition. In addition we suggest that administration of T-4 to the hypothyroid fetus in utero is an acceptable modality of treatment and may help to minimize irreversible mental retardation in known high risk infants. However, further studies are necessary to assess the effectiveness and safety of this approach.


A cycle of plasma testosterone in the human male. The object of the study was to assess the lability of testosterone levels in plasma of normal human males over a long period of time and to search for periodicities in changing levels. Blood samples obtained from 20 healthy young men every second day for 2 months were assayed for total testosterone concentration by radioligand saturation analysis with late-pregnancy plasma. The flucturations of plasma testosterone levels over the total time span were substantial for most individuals; the coefficients of variation ranged from 14 to 42% (median 21%). The presence of periodic functions in these fluctuations was tested by 4 different, relatively independent methods. Close agreement among at least 3 analytic methods was found for 12 out of the 20 subjects. These 12 subjects had cycles of plasma testosterone levels with periods ranging between 8-30 days, with a cluster of periods around 20-22 days. The majority of such cycles were significant at least at the 5% level. The mean amplitudes of these cycles ranged from 9 to 28% of the subjects' mean testosterone levels (average 17%).


Spontaneous fluctuations of human placental lactogen during normal pregnancy. Six women in the 3rd trimester of normal pregnancy had measurements of circulating placental lactogen (hPL) levels using a continuous blood sampling technique for 10-15 h. In addition, in 3 pregnant women hPL was assayed at 10-min intervals for 60-90 min. Both these procedures showed that hPL serum levels fluctuate irregularly during normal pregnancy. The magnitude and frequency of these fluctuations make the significance of a single hPL determination less reliable as a test of placental function.


Circulating estradiol, estrone and gonadotropin levels following the administration of orally active 17beta-estradiol in postmenopausal women. Ingestion of a single tablet containing 2 mg micronized 17beta-estradiol (E-2) produced marked increases in the serum concentrations of E-2 and estrone (E-1) in 9 postmenopausal women. The rise in circulating E-2 became significant within 2 h, reached a maximum (110 pg/ML; 437% increase) at 5 h, and remained significantly elevated at 8 h posttreatment. By 24 h, the serum E-2 concentration was not significantly different than baseline. In contrast, a more rapid (within 1 h) and pronounced (4-fold) increase in the serum concentration of E-1 was observed. This rise continued until a peak (467 pg/ml; 2000%) was reached 6 h posttreatment. Thereafter, the serum E-1 concentration declined progressively but was still significantly elevated (140 pg/ml; P smaller than 0.01) 24 h after treatment. Serum concentrations of FSH AND LH were significantly decreased within 6 and 3 h, respectively and both gonadotropins remained significantly suppressed 24 h following the ingestion of E-2. The ratios of circulating E-1: E-2 reported herein (ca. 3-6) were much higher than those observed by other investigators following iv E-2 (I.E., smaller than 1). Thus the data indicate that micronized E-2 peros is readily absorbed and that during this process a significant portion of the hormone is converted to E-1 by the gstrointestinal tract. In addition, 2 mg oral E-2 exerts significant biologic activity as assessed by serum gonadotropin suppression.


Metyrapone test in Cushing's disease.

Metyrapone test in Cushing's disease. The urinary 17-hydroxycorticosteroid response to 2 g of metyrapone given orally at 10 PM was compared with that following the standard test in which 750 mg of metyrapone was given at 4 hourly intervals for 6 doses. Both tests were performed on four occasions in 3 patients with Cushing's disease. Increments in urinary 17-hydroxycorticosteroid excretion during the modified test were 7.0, 7.5, 8.4 and 23.3 mg/day, whereas with the standard test, increments ranged from 29.5 to 56.8 mg/day. The urinary 17-hydroxycorticosteroid response to the 2 g dose of metyrapone at 10 PM was marginal in 3 of the 4 studies. Urinary 17-hydroxycorticosteroid excretion with the modified metyrapone test varied from 10.7 to 44% of that found with the standard test. Since urinary steroid excretion may vary considerably in patients with Cushing's syndrome as was evident in 2 of the 3 patients studied, the data suggest that the modified metyrapone test should not be used in preference to the standard test in evaluating Cushing's syndrome. It appears that the modified test could lead to erroneous conclusions.


Plasma renin activity in normal subjects from infancy to puberty. Immunoassayable Plasma renin activity (PRA) was measured in normal subjects aged from 15 days to 15 years. A very progressive decrease of PRA with age could be demonstrated. Practical conditions for the study of PRA were defined.


Plasma glucagon and alanine responses to acute insulin deficiency in man. Plasma glucagon levels rose within 1 hr after withdrawal of insulin in 7 juvenile-type diabetics previously kept normoglycemic by prolonged intravenous infusions of insulin. These changes preceded subsequent elevations in plasma alanine levels. Individual rises in plasma glucagon were correlated with elevations in plasma glucose, beta-hydroxybutyrate, free fatty acid, and glycerol levels, suggesting that glucagon may play an important role in the development of diabetic ketoacidosis in man.


Early variations of plasma somatomedin activity in the newborn. In neonates, plasma somatomedin as measured by the porcine cartilage assay was very low during the first day of life. A striking increase was observed on day 4 and 5, with a return to lower values at a later age. These findings indicate an early capacity to generate somatomedin activity in newborns.


Appearance of hCG in pregnancy plasma following the initiation of implantation of the blastocyst. Plasma chorionic gonadotropin levels were measured by three different assay methods during early pregnancy in four patients following induction of ovulation with Pergonal and hCG. Radioligand-receptor assay of unextracted samples was subject to non-specific interference by plasma proteins, causing an apparent elevation of gonadotropin levels during the first few days after fertilization. By contrast, the gonadotropin values measured by a highly sensitive LH/hCG bioassay were consistent with those obtained with a sensitive and specific radioimmunoassay for hCG, and showed that the first significant rise in plasma hCG occurred 9 to 13 days after ovulation. These results indicate that hCG does not appear in the maternal circulation until after the initiation of implantation of the blastocyst.


Demonstration of insulin resistance in untreated adult onset diabetic subjects with fasting hyperglycemia. We have used a continuous intravenous infusion of glucose (6 mg/kg/min), insulin (80 mU/min), epinephrine (6 mug/min), and propranolol (0.08 mg/min) to directly assess insulin resistance in 14 untreated adult onset diabetics with a mean (plus or minus SE) fasting plasma glucose level of 217 plus or minus 17 mg/100 ml. During the infusion endogenous insulin secretion is inhibited and steady-state plasma glucose and insulin levels are achieved after 90 min. Since similar steady-state levels of plasma insulin are achieved in all subjects, the plasma glucose concentration observed during the steady-state period is a measure of an individual's insulin resistance. Under these conditions, the mean (plus or minus SE) steady-state plasma glucose level of the 14 diabetic patients was 350 plus or minus 16 mg/100 ml, while that of 12 normal subjects was 121 plus or minus 4 mg/100 ml. Additional studies were performed in which control subjects and patients with diabetes had their fasting plasma glucose levels acutely raised or lowered to comparable levels before receiving the basic infusion mixture of glucose, insulin, epinephrine, and propranolol. The results of these studies indicated that differences in initial plasma glucose levels could not account for the different glucose responses of the two groups to the basic infusion. Finally, the mean (plus or minus SE) steady-state plasma glucose level of 104 plus or minus 17 mg/100 ml observed during the same basic infusion in five patients with fasting hyperglycemia (mean plus or minus SE, 142 plus or minus 12 mg/100 ml) secondary to chronic pancreatitis suggested that neither chronic hyperglycemia nor hypoinsulinemia per se necessarily lead to insulin resistance. These results demonstrate that marked insulin resistance exists in adult onset diabetics with fasting hyperglycemia. Since previous studies have documented the presence of insulin resistance in patients with chemical diabetes, the possibility exists that insulin resistance may be characteristic of adult onset diabetes mellitus.


Biosynthesis of bile acids in man. Hydroxylation of the C27-steroid side chain. The first step in the degradation of the steroid side chain during biosynthesis of bile acids from cholesterol in man was studied in microsomal and mitochondrial fraction of homogenate of livers from 14 patients. The microsomal fraction was found to catalyze an efficient 25-hydroxylation of 5,8-cholestane-3a,7a,12atriol. A small extent of 23-, 24-, and 26-hydroxylation of the same substrate was observed. 53-Cholestane-3a,7adiol was hydroxylated in the 25-position only to a very small extent. The mitochondrial fraction was found to catalyze 26-hydroxylation of cholesterol, 5-cholestene-3P,7a-diol, 5P-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one, and 5,0-cholestane-3a,7a,12a-triol. Addition of Mg++ stimulated the 26-hydroxylation of cholesterol but had no effect or an inhibitory effect on 26-hydroxylation of the other substrates, indicating a heterogeneity of the mitochondrial 26-hydroxylating system. The level of 26-hydroxylase activity towards different substrates varied considerably with different mitochondrial preparations. The roles of the microsomal and mitochondrial 26- hydroxylations as well as the microsomal 25-hydroxylation in biosynthesis of bile acids in man are discussed. The results indicate that microsomal 26-hydroxylation is less important than mitochondrial 26-hydroxylation under normal conditions. The possibility that microsomal 25-hydroxylation is important cannot be ruled out.


The choleretic effect of iodipamide.

The choleretic effect of iodipamide. It is well established that a number of organic anions are excreted by the liver into bile in association with a marked increase in bile flow. Previous studies have shown that iodipamide (3,3'-(adipoyl-diimino)bis[2,4,6-triiodobenzoic acid]), the radiographic contrast material used for intravenous cholangiography, is a potent choleretic. Experiments were performed in unanesthetized dogs to determine if the increased bile flow produced by iodipamide is canalicular or ductular in origin, to quantitate the choleresis associated with iodipamide and taurocholate excretion, and to correlate these findings with the results of in vitro studies in which the osmotic activities of iodipamide and taurocholate in both isotonic saline and bile were determined. The plasma erythritol clearance increase linearly with the excretion of iodipamide, indicating that iodipamide stimulates canalicular bile flow. The choleretic potency of iodipamide (22 ml/mmol) is approximately 3 times that of taurocholate (7.8 ml/mmol), yet the osmotic activity of iodipamide in bile (1.5 mosmol/mmol) is only twice as great as that of taurocholate in bile (0.8 mosmol/mmol). It therefore appears that, per unit of effective osmotic solute secreted, iodipamide carries more water into the bile canaliculi than does taurocholate.


Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal--leukocyte interaction. Since oxygen-free polymorphonuclear neutrophils (PMN) cannot kill Staphylococcus aureus normally, the usual mechanisms for PMN bactericidal activity probably involve hydrogen peroxide or superoxide. Catalase can destroy hydrogen peroxide, and superoxide dismutase breaks down superoxide. Experiments were performed to study the influence of these enzymes (which are found in staphylococci) on virulence for mice or on leukocyte-bacterial interaction. 15 staphylococcal strains were injected i.p. into mice to quantitate virulence. There was good correlation between staphylococcal catalase activity and mouse lethality (r equals 0.88) but no correlation between staphylococcal superoxide dismutase activity and mouse lethality (r equals 0.14). Exogenous catalase (10,000 U/ml) increased the virulence of low-catalase staphylococci, but exogenous superoxide dismutase (200 mug/ml) did not alter the virulence of staphyloccal strains. C14=labeled high-catalase or low-catalase staphylococci were ingested equally well by PMN, with or without the addition of exogenous catalase. A high-catalase staphylococcal strain was killed relatively poorly by PMN, and addition of exogenous catalase (but not superoxide dismutase) decreased the ability of PMN to kill a low-catalase strain. Iodination of bacterial proteins by PMN is related to hydrogen peroxide, and a high-catalase staphylococcal strain was iodinated only 63% as much as a low-catalase strain. Addition of exogenous catalase decreased iodination of the low-catalase strain by 23%. These findings suggest that staphylococcal catalase protects intraphagocytic microbes by destroying hydrogen peroxide produced by the phagocyte. Thus, catalase may be a significant staphylococcal virulence factor.


Effect of increased peritubule protein concentration on proximal tubule reabsorption in the presence and absence of extracellular volume expansion. The effect of increased peritubule capillary oncotic pressure on sodium reabsorption by the proximal tubule of the dog was investistigated after extracellular volume expansion (ECVE) with Ringer's solution or during continued hydropenia. Control measurements were made after ECVE or during hydropenia and again during renal arterial infusion with hyperoncotic albumin solution. Absolute reabsorption by the proximal tubule was calculated from fractional reabsorption and single nephron filtration rates as determined by micropuncture. Direct measurements of efferent arteriole protein were used to determine efferent arteriolar oncotic pressure. Albumin infused into the renal artery after ECVE significantly increased efferent oncotic pressure by 17.6 plus or minus 5.3 mm Hg. Fractional and absolute reabsorption by the proximal tubule increased from 20 plus or minus 6 to 37 plus or minus 5% and from 22 plus or minus 6 to 36 plus or minus 7 nl/min, respectively. During hydropenia, the albumin infusion significantly increased efferent oncotic pressure by 15.0 plus or minus 4.4 mm Hg. However, in contrast to the effect seen during ECVE, neither fractional nor absolute reabsorption was changed, delta equals 0.3 plus or minus 1.5% and 3 plus or minus 5 nl/min, respectively. Single nephron filtration rates were not significantly different between the groups and were unchanged by the albumin infusion. Peritubule capillary hydrostatic pressures, measured with a null-servo device, were not changed by the albumin infusion in either group. Renal interstitial hydrostatic pressure, measured from chronically implanted polyethylene capsules, was decreased significantly from 7.2 plus or minus 0.9 to 3.4 plus or minus 0.6 mm Hg in the hydropenic group and from 0.6 plus or minus 0.6 to 4.8 plus or minus 0.7 mm Hg in the Ringer's expanded group. In the hydropenic group, the increase in efferent oncotic pressure was nearly compensated for by changes in interstitial forces so that the calculated net force for capillary uptake was almost unchanged, 17.8 mm Hg before vs. 21.4 mm Hg during the albumin infusion. The increased efferent oncotic pressure in the Ringer's expanded group was not compensated, so that the calculated net force for uptake was increased, 11.9 mm Hg before to 22.2 mm Hg during the albumin infusion. Thus, while the increase in efferent oncotic pressure during albumin infusion was not significantly different between the groups, absolute and fractional reabsorptions were increased only in the animals in which the extracellular volume was expanded. The results suggest that ECVE alters the effect of increased peritubule oncotic pressure on sodium reabsorption by the proximal tubule.


Platelet hypersensitivity induced by cholesterol incorporation. Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating atents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] equals 1.0, "cholesterol rich" (C/P eauals 2.2), or "cholesterol poor" (C/P equals 0). Cholesterol-normal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 37 degrees C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was three-fold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P less than 0.001) and 15-fold increase to ADP aggregation (P less than 0.001), as determined both by aggregometry and by [13C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. These studies demonstrate that the cholesterol content of platelets is dependent on the lipid composition of the milier. Cholesterol acquired by platelets may exert its effect on platelet function by a modification of the platelet membrane.


Orthophosphate transport in the erythrocyte of normal subjects and of patients with X-linked hypophosphatemia. We have examined the mechanism of TCA-soluble orthophosphate (Pi) transfer across the membrane of mature human erythrocytes in normal subjects and in patients with X-linked hypophosphatemia (X-LH). The studies were carried out largely at pH 7.4 and 37 degrees C, in partial stimulation of conditions in vivo. (a) At physiological concentrations (1-2 mM) Pi enters the intact normal erythrocyte down its chemical gradient and under no conditions could we identify a steady-state trans-membrane gradient for Pi greater than 0.6. Calculations of the phosphate anion distribution ratio using the Nernst equation yield theoretical values that closely approximate observed values. (b) Glycolytic inhibitors have little effect on total entry of 32Pi inti erythrocytes but they do affect the intracellular distribution of Pi. In the presence of iodoacetamide, label accumulates almost exclusively in the orthophosphate pool and less than 1% enters the organic phosphate pool. (c) Specific activity measurements in unblocked cells indicate that Pi anion equilibrates first with its intracellular Pi pool. These initial findings imply that neither group translocation, nor energy coupling, influence Pi permeation into the human erythrocytes. (d) The relationship between 32P entry and extracellular Pi concentration is parabolic in the presence of chloride, and linear in the presence of sulfate. The kinetics of concentration dependent entrance cannot be examined and saturability of Pi entry cannot be identified under these conditions. (e) The competitive inhibitor arsenate partially inhibits the initial rate and steady-state flux of orthophosphate in erythrocytes treated with iodoacetamide to inhibit glycolysis. However, a significant portion of Pi transport escapes arsenate inhibition. (f) Activation energies for Pi entry, in nonglycolizing erythrocytes are much higher than those required by simple diffusion in an aqueous system. (g) Neither the inward or outward movement of Pi is modulated by trans-phosphate. These latter findings suggest that transport of phosphate across the human erythrocyte is compatible with slow facilitated diffusion with symmetry for influex and efflux. The transmembrane chemical distribution ratio, and the equilibrium flux of Pi were not different from normal in the X-LH erythrocyte. Nor did the extracellular Pi concentration, arsenate, or temperature affect Pi entry differently in the two types of cells. We dedjce that different gene products serve the diffusional type of Pi transport in the erythrocyte membrane and the saturable component of transepithelial absorption in the gut and kidney. Only the latter is affected by the X-LH mutation. The former is apparently present not only in erythrocytes but also in epithelial tissue, where it can serve the absorption of pharmacologic amounts of Pi in the therapeutic repair of the depleted phosphate pools in X-LH.


Conversion of human erythrocyte-adenosine deaminase activity to different tissue-specific isozymes. Evidence for a common catalytic unit. Adenosine deaminase activity resides in various characteristic isozymes in red blood cells (RBC-ADA) and other tissues. Absence of RBC-ADA has been reported in a proportion of patients with autosomally inherited severe combined immunodeficiency (SCID). We have previously reported that the tissue isozymes of ADA are also deficient in children with SCID and RBC-ADA deficiency, although these isozymes differ from RBC-ADA in molecular weight, accessible SH groups, and electrophoretic mobility. The deficiency of all types of ADA in SCID implies that a catalytic unit of ADA in each isozyme is coded by the same structural gene. The relationship of RBC-ADA and the different tissue ADA isozymes is the subject of this paper. Incubation of RBC-ADA with ADA-deficient liver, kidney, and fibroblast extracts resulted in the appearance of new isozymes of ADA. These newly generated isozymes had the physicochemical and electrophoretic characteristics of the tissue-specific isozymes obtained from normal tissues. The electrophoretic mobility of the isozyme generated appeared to depend upon the tissue utilized and corresponded to the electrophoretic mobilities of the ADA isozymes found naturally in each of the different tissues. Additionally, the genetically determined polymorphism exhibited by RBC-ADA could be detected in the isozyme generated. Incubation with normal kidney also caused conversion of the RBC isozyme to the kidney form. These findings further support the concept that the catalytic activity of each of the several forms of the ADA enzyme resides in a single molecule coded at the same genetic locus as is defective in one form of SCID. The tissue-specific isozymes, which differ in electrophoretic mobility and molecular weight, are generated by interaction of the RBC catalytic unit with tissue-specific factors present in the different tissues of normal humans and patients.


Restoration by purified C3b inactivator of complement-mediated function in vivo in a patient with C3b inactivator deficiency. In a patient with lifelong increased susceptibility to infection and multiple abnormalities in complement-mediated functions, the infusion of normal plasma had been seen to produce a prolonged partial correction of serum abnormalities. It was subsequently shown that the patient was genetically deficient in the C3b inactivator and that immunochemical depletion of C3b inactivator from normal serum resulted in abnormalities similar to those found in the patient's serum, including alternative pathway C3 activation. Highly purified C3b inactivator was obtained from the euglobulin fraction of normal human serum, sterilized by filtration, and infused intravenously. Partial or complete correction of almost all the known serum abnormalities was obtained. C3b almost disappeared from the serum within 4-5 h, as did Factor C activity. Native C3, C5, and serum hemolytic activity rose to normal or near-normal levels over 4 days and were sustained for another week. Factor B, properdin, opsonic activity, and bactericidal activity reached a level at least two-five times that found before the infusion within 24 h and fell over the next 5 days. These observations prove the primary role of C3b inactivator deficiency in the patient's disease and demonstrate clearly the curcial role in vivo of C3b inactivator in modulating alternative pathway activity.


Molecular weight profile of fat globule membrane proteins. Fat globule membrane material, isolated by churning cream that had been washed four times, was extracted sequentially with .6 M potassium chloride and centrifuged to yield pellet and supernatant fractions. Compositional data indicated that lipid components were removed preferentially into the supernatant fractions. Electropherograms of the pellet and supernatant fraction in 10% and 5% sodium dodecyl sulfate-containing polyacrylamide gels revealed that the protein species originally present in the membrane associated into higher molecular weight species concomitant with the removal of lipid moieties. Thus, a contribution of the lipid moiety to membrane integrity is suggested. Approximately 17 protein-stained zones, ranging in molecular weight from 13,500 to 208,000 daltons, were observed in gels of the membrane material.


Effects of insulin upon fatty acids synthesis from pyruvate, lactate, and glucose in rat mammary cells. In isolated rat mammary secretory cells, insulin stimulated fatty acid synthesis from pyruvate three times, stimulated glucose conversion to fatty acids 1.2 to 1.5 times, and decreased lactate conversion to fatty acids 20 to 30%. Incubation of glucose and pyruvate together depressed fatty acid synthesis from glucose not attributable to isotope dilution. Glucose stimulated conversion of pyruvate-2-14 carbon to fatty acids without significantly affecting pyruvate-1-14 carbon conversion to 14-carbon dioxide. At differing concentrations, the electron acceptors phenazine methosulfate and N,N,N',N'-tetramethyl-p-phenylene-diamine alleviated the depression by insulin of lactate conversion to fatty acids. The data support concepts that: (1) insulin acts at important sites other than or in addition to glucose transport in regulating mammary secretory cell metabolism and, particularly, fatty acid synthesis; (2) insulin actions upon fatty acid synthesis can vary dependent upon cellular redox state (insulin increases fatty acid synthesis in cells with a low redox state and decreases fatty acid synthesis in cells in a very reduced state); and (3) pyruvate depresses glucose carbon flux through the Embden-Meyerhof pathway.


Epinephrine inhibiting milk ejection in lactating cows. Blood oxytocin was determined on 10 Holstein cows during normal milking and during milking when epinephrine was injected intravenously before or after udder stimulation. The average peak oxytocin concentration during normal milking was 399.7 muU/ml plasma and was reached at 1 min after teat cup application. Oxytocin concentration in blood plasma declined rapidly after peak concentration and dropped to 30.5 muU/ml plasma within 4 min after the start of milking. By 5 min after removal of the teat cups, it had declined to less than 4 muU/ml plasma. The administration of epinephrine, either before or after udder washing, inhibited milk ejection as indicated by milk production and oxytocin concentrations. Peak oxytocin concentrations were 1.05 muU/ml plasma 1 min after teat cup application when epinephrine was injected before udder washing and 8.6 muU/ml plasma at teat cup application when epinephrine was injected after udder washing. These results and the use of a beta receptor blocker to inhibit the effect of epinephrine at the myoepithelial cell level indicated that epinephrine inhibited release of oxytocin from the neurohypophysis.


Feeding following intrahypothalamic injections of calcium and magnesium ions in sheep. To study ionic effects on hypothalamic control of feeding, seven sheep were each prepared with six bilateral medial-hypothalamic guides. Hypothalamic loci were identified where feeding resulted following 1 mu 1 injections of calcium chloride and/or magnesium chloride. Doses of 1 and 2 mumol were most effective to induce feeding, and potassium chloride was not effective. Injections of phentolamine, LB-46, carbachol, atropine, or prostaglandin E1 did not inhibit the feeding responses following magnesium chloride injections into magnesium ion responsive loci. Feeding following calcium chloride injections into calcium ion responsive loci was blocked by atropine and reduced by phentolamine. Carbachol and prostaglandin E1 elicited feeding when injected into magnesium but not calcium ion responsive loci. Injections of carbachol followed by magnesium chloride resulted in greater feeding than when either chemical was injected alone. Calcium and/or magnesium ion bound feeding loci were in the anterior, ventromedial, dorsomedial, posterior and lateral hypothalamus, and the mammillary bodies. These ions elicited feeding in sheep perhaps by decreasing excitability of feeding-inhibitory neural pathways in the hypothalamus.


Effect of glucose load on synthesis of plasma glucose in lactating cows. We assess the effect of an intravenous glucose load on production of glucose in lactating dairy cows. Isotope dilution techniques were utilized to measure transfer rate of plasma glucose (with tritiated glucose) and gluconeogenesis (with carbon-14 labeled propionate or carbon dioxide). A glucose load equivalent to the cow's normal transfer rate decreased endogenous glucose output two-thirds and decreased gluconeogenesis by half. Glucose concentration and transfer rate were higher within 2 h after glucose load started while carbon-14 transfer to plasma glucose became increasingly depressed and persisted for at least 1 h after loading terminated. Reduced plasma glucose synthesis from propionate during loading was associated with increased transfer of propionate carbon to plasma phospholipids, triglycerides, and cholesterol esters, and with increased plasma insulin concentration. It appears that glucogenic precursors are utilized for greater synthesis of lipid (and presumably glycogen) and that loading leads to metabolic changes causing net reduction in gluconeogenic capacity. These changes could have important implications for use of intravenous glucose in treating hypoglycemic metabolic conditions.


Effects of zinc, copper, and manganese supplementation of high-concentrate ration on digestibility, growth, and tissue content of Holstein calves. The effects of supplemental zinc, copper, and manganese alone or in combination in a high-concentrate ration were studied in 16 Holstein bull calves during a 10-wk feeding trial. Metabolism was studied after the feeding trial. Apparent digestibility of dry matter, nitrogen, and gross energy, and nitrogen retention and urinary excretion of zinc, copper, and manganese were determined. The calves were slaughtered after the experiment, and liver, heart, and kidney were taken for analyses of trace minerals. Supplementation of the basal ration with the trace minerals did not affect body weight gains, which averaged 1.42 kg daily. The addition of trace minerals did not affect apparent digestibility co-efficients. A zinc-manganese interaction in digestion of nitrogen and gross energy was significant. Higher dietary manganese caused increased zinc concentrations in the liver, kidney, and heart. The copper concentration of liver was decreased by dietary zinc and increased by dietary copper and manganese. Supplemental manganese increased its net retention. There was no evidence of deficiency of any trace minerals in the unsupplemented treatments.


Combinations of glycerol percent, glycerol equilibration time, and thawing rate upon freezability of bull spermatozoa in plastic straws. Twelve ejaculates were used in a central composite experiment to test 15 combinations of glycerol (7, 9, 11, 13, or 15%), glycerol equilibration times (1, 2, 4, 8, or 16 h) and thawing rates (water at 35 C for 15 s, 50 C for 13 s, 65 C for 11 s, 80 C for 9 s, or 95 C for 7 s). Semen was diluted in heated skim milk-glycerol, packaged in .3-ml. Continental U.S. straws and frozen in liquid nitrogen vapor. Based on post-thaw progressive sperm motility after storage at -196 C for 9 to 11 days, estimated optima from multiple regression were 10.7% for glycerol, 2.0 h for glycerol equilibration time, and 76 C for thawing bath temperature. Only the linear effect for each variable was significant. Much faster thawing rates and shorter glycerol equilibration times than those for freezing bull spermatozoa in glass ampules should be used for maximum post-thaw sperm motility in straws.


Effect of sperm numbers on fertility of frozen bull spermatozoa in skim milk diluent. Insemination doses of 10, 15, and 20 million progressively motile spermatozoa prior to freezing were compared in a split-ejaculate fertility experiment involving 4,603 first service inseminations to 34 semen samples from six Angus bulls. Based on 60- to 90-day nonreturn rates, fertility was significantly lower for inseminating doses of 10 and 15 times 10-6 than 20 times 10-6 motile sperm (61, 62, and 70%). The 1-ml doses of skim milk diluted semen were frozen in glass ampules. The post-thaw number of motile sperm per ampule after freezing and storage for 3 wk in liquid nitrogen averaged 5.5, 8.4, and 12.4 times 10-6 for corresponding pre-freeze numbers of 10, 15 and 20 times 10-6 motile sperm/ml and ranged from 4.0 to 15.7 times 10-6/ml. Frozen semen packaged in ampules should contain more than 8.4 times 10-6 motile cells post-thaw per ampule.


Genetic and environmental effects on serum cholesterol of dairy cattle of various ages. Serum cholesterol of dairy cattle, particularly when measured on animals of the same approximate age, is a heritable trait. Heritabilities were .50 for first lactation cows and .19 for heifers from 2 to 22 mo of age. Serum cholesterol increases with increasing age. Serum cholesterol was 192.8 mg/100 ml in cows and 121.4 mg/100 ml in heifers. Serum cholesterol was relatively low at the onset of lactation, increased at mid-lactation, and leveled off or decreased in late lactation. Month of calving, year of sampling, and ration did not influence serum cholesterol of first lactation cows. However, variation in both month and year of sampling was significant for heifers.


Prevention of parturient paresis by a low-calcium diet prepartum: a field study. Twenty of 60 control cows fed a "normal" diet prepartum developed parturient paresis whereas none of 37 fed a low-calcium (13 to 18 g/cow per day) diet prepartum developed the disease.


Dietary survey of low-income, rural families in Iowa and North Carolina. I. Research procedures. Methods used in assessing the dietary intake of selected low-income, rural families are described. The sample consisted of 668 families randomly selected from predetermined locations in Iowa and North Carolina. A 24-hr. recall method supplemented with food models was used in gathering data on family food intake. The nutritional "adequacy" of the family diets was then evaluated according to family composition and the 1968 Recommended Dietary Allowances. The adequacy of individual nutrients was also evaluated by the same criteria. Nutrients studied included: protein, calcium, iron, vitamin A, ascorbic acid, thiamin, and riboflavin. The selection of the method in relation to opinions of previous investigators is discussed.


Dietary survey of low-income, rural families in Iowa and North Carolina. II. Family distribution of dietary adequacy. Distribution patterns of Iowa and North Carolina families for dietary adequacy at various income levels were compared according to: (a) two family dietary standards, based on the 1968 Recommended Dietary Allowance and the Ten-State Nutrition Survey dietary standards, and (b) two income standards, family income and per capita income. A 24-hr. recall method was used to collect the dietary data. Nutrients studied included: protein, calcium, iron, vitamin A, ascorbic acid, thiamin, and riboflavin. Clear differences emerged in family distribution patterns by the two dietary standards and by the two income standards. Comparison of family distribution patterns for dietary intake by the two standards showed that percentages of families with poor nutrient intakes were lower by Ten-State evaluation than for the recommended allowance evaluation, except for protein and iron. For example, the percentage of families with poor calcium intakes, by Ten-State criteria, was approximately half that measured by the recommended allowances. While, according to the latter, calcium was the most limiting nutrient in many family diets, vitamin A was most limiting by Ten-State evaluation. The proportions of families with poor ascorbic acid, thiamin, and riboflavin intakes were also lower by Ten-State standards. While the iron adequacy remained approximately the same by both dietary standards, the percentage of families with poor protein intakes was higher by the Ten-State criteria. A higher percentage of families at each income level had fair and good diets by Ten-State comparison. Family distribution patterns for intakes of individual nutrients at various family income levels demonstrated a positive relationship between nutritional intake and income. Proportions of families with poor nutrient intakes according to per capita income tended to increase with the income level. For both income standards, the percentages of families with good and fair diets in the total family sample gradually increased with income. Percentages of Iowa families with fair and good diets at various income levels were, in general, higher than those of North Carolina families. The lowest and the highest percentages of families with poor diets of two population groups were higher for per capita income distribution than for family income distribution.


Dietary survey of low-income, rural families in Iowa and North Carolina. III. Contribution of food groups to nutrients. Percentage contributions of specific food groups indicated in the Basic 4 to protein, calcium, vitamin A, and ascorbic acid in one day's diets of thirty-five Iowa families and twenty-five North Carolina families are reported. All members of the families ate all meals at home. Their diets met or exceeded two-thirds of the 1968 Recommended Dietary Allowances for seven nutrients. Data were collected by the 24-hr. recall method. While the percentage contribution of the meat group to protein intake of the North Carolina families ranged from zero to 84 per cent, that of the Iowa group was from 35 to 85 per cent. The range for the percentage contribution of the milk group to calcium intake of North Carolina families was similar; however, in the Iowa group the range was from 2 to 97 per cent. The vitamin A-rich fruit and vegetable group contributed from zero to 99 per cent for the North Carolina families of vitamin A intake and from zero to 88 per cent for the Iowa group. Ascorbic acid-rich fruits and vegetables contributed from zero to 100 per cent of the ascorbic acid intake of the Iowa group and from zero to 95 per cent in North Carolina families. The results support the argument that the exclusive use of the "Daily Food Guide" or the "Basic 4" to assess nutritional intake of diets may produce biased in formation.


Urinary calcium excretion in relation to kidney function in the adult. A study based on 55 hospital patients (age groups 40-59, 60-69, 70-79) demonstrated a significant correlation between the glomerular filtration rate (as expressed by endogenous creatinine clearance) and the mean urinary calcium concentration. Excluded were patients with thyroidal or hypophyseal disorders, osteoporosis, renal stones immobilizing conditions, or those treated with corticosteroids. The study patients had a normal fluid intake and did not receive any diuretics. The urinary calcium concentration test is less laborious and less liable to laboratory errors than the creatinine clearance test, which requires a 24-hour urine collection. Because of the gradual decrease in the glomerular filtration rate in the elderly, this test should be of value in geriatric practice.


Pulmonary infections in elderly patients: treatment with cefazolin. The effects of cefazolin were studied in 15 hospital patients (average age, 67) with respiratory-tract infections. The infections were chiefly bronchopneumonia caused by organisms such as D. pneumoniae, Staph. aureus, or E. coli. In 13 of the 15 patients the results were excellent to good (eradication of the primary pathogen and clinical improvement); in the other 2 patients the response was fair. Intramuscular injection was well tolerated and only one patient complained of pain at the site of injection; no other adverse clinical effects were observed. In the laboratory determinations, including liver and kidney function tests, the only abnormalities were 2 cases in which the Coombs' test gave positive results without hemolysis. Cefazolin seems to be a safe and effective antibiotic for the treatment of respiratory-tract infections caused by cefazolin-susceptible organisms.


Prevalence of hypertension among the elderly in an urban community. The results of a hypertension screening project conducted in senior citizen agencies in Manhattan are presented. The prevalence of hypertension among elderly females tended to conform to the data of the Nation Health Surgery of 1960-62, but the prevalence among elderly males was much higher than in the National Survey. The prevalence of hypertension varied inversely with the educational level. Various implications for future medical evaluations of the elderly are discussed, based on the clinical and social data obtained.


Important factors in the examination and care of old patients. The author emphasizes the importance of certain factors in the initial assessment of geriatric patients. The geriatrician should be a keen observer, and not neglect simple tests. Thus diagnostic mistakes will be kept to a minimum and he will be rewarded by the improved welfare of his patients.


Differential permeation of Artemia cysts and cucumber seeds by alcohols. The rate of penetration of the simpler alcohols into brine shrimp cysts and cucumber seeds was studied. In solutions below 70% the rate of penetration is related to lipid solvent capacity of the alcohol. In concentrations above 70%, particularly in absolute alcohols, methanol penetrates brine shrimp rapidly and ethanol penetrates slowly. All the other alcohols tested did not pepetrate the dormant structures. Ethionine and deuteroxy-methanol did not affect the rate of penetration of methanol. It is suggested that in dehydrated membranes the lipid moiety is protected by a continuous sheet of protein. Methanol, which is fairly similar to water, is probably able to penetrate the membrane by initiating a conformation change in the protein, exposing the lipid which subsequently dissolves in the methanol thus destroying the membrane.


Rapid chemical dehydration of samples for electron microscopic examinations. Acidified 2,2-dimethoxypropane was used to chemically dehydrate biologic tissues for examination in the electron microscope. The ultrastructural integrities of single-celled algae, plant tissues (cotyledon, root, leaf) and animal tissues (liver, pancreas, muscle, cartilage) were maintained. Our technique was simpler and quicker than physically exchanging water for organic solvents (e.g., acetone, ethanol) as generally performed in microscopy.


Histochemical demonstration of mitochondrial dehydrogenases in developing normal and nervous mutant mouse Purkinje cells. The mouse mutation nervous (nr), expressed as a recessive, causes a morphologic change in cerebellar Purkinje cell perikaryonal mitochondria during the 2nd postnatal week which is followed by the selective degeneration of most Purkinje cells. In the present study, mitochondrial dehydrogenase activities (succinic acid dehydrogenase, lactic acid dehydrogenase, malic acid dehydrogenase, reduced diphosphopyridine nucleotide diaphorase and succinic semialdehyde dehyrogenase) are found by light microscopy to be similar in the perikarya of normal and nervous Purkinje cells until after mitochondrial rounding has begun to occur, but then staining increases markedly in nervous. Although only a small fraction of mitochondrial functions were examined, those enzymes assayed were present and competent. In a sense, the only mitochondrial abnormalities demonstrated thus far in nervous Purkinje cells are their unusual shape and probable loss of motility.


Characterization of chemotactic activity produced in vivo by a cell-mediated immune reaction in the guinea pig. A system that allows repeated sampling of peritoneal fluid at various time intervals has been adapted to study mechanisms of leukocyte accumulation in vivo. Application of this technique in guinea pigs exhibiting delayed hypersensitivity (DH) to horse radish peroxidase (HRPO) has allowed characterization of some events after i.p.challenge with the sensitizing antigen. Within 24 hr of the administration of HRPO i.p. to such animals there is a significant increase in the number of peritoneal macrophages and in the chemotactic activity (CTX) for macrophages in the sampled peritoneal fluid. At 48 and 72 hr the CTX returns to the pre-challenge level and i.p. macrophages appear to be actively phagocytic. Molecular sieve chromatograms of concentrated peritoneal fluid obtained 24 hr after i.p. challenge with HRPO and of supernatants derived from immune spleen cells cultured in the presence of HRPO in the presence of HRPO in vitro revealed that the major portion of CTX for homologous macrophages eluted in the region of the 12,500 dalton protein marker. The partially purified CTX obtained from peritoneal fluid and supernatants of spleen cell cultures was heat stable (56 degrees C for 30 min) and was destroyed by trypsin digestion. These data demonstrate that a chemotactic factor (LDCF) obtained in vitro, is present in vivo at the site of a cell-mediated immune reaction. Moreover, these observations demonstrate the feasibility of studying the kinetics of leukocyte accumulation and the production of mediators of inflammation at the site of well defined immunologic reactions in vivo.


Quantitative comparison of techniques used to measure complement-mediated cytotoxicity of nucleated cells. ?The cytotoxicity of nucleated cells by specific antibody and complement can be guantitated in vitro by several methods. Trypan blue exclusion, 51-Cr release, inhibition of uptake of 3-H-thymidine, and inhibition of colony formation are the four assays that we used to guantitatively compare the C-mediated cytotoxicity of Chinese hamster lung (CHL) cells. CHL cells were sensitized with either guinea pig or rabbit anti-CHL antisera, and exposed to guinea pig, human, or rabbit C. We found that the relative activities of the antibodies and the complements were not dependent on the method of measurement of cytotoxicity, i.e., a given  pair of antiserum-complement were not dependent on the method of measurement of cytotoxicity, i.e., a given antiserum-complement titer, regardless of the assay used to measure cytotoxicity. However, the titers of the different antibody-complement pairs differed from each other.


The properities of lymphocytes which carry immunologic memory of phiX 174. Cells that carry immunologic memory of phiX 174 have been studied in the rat. Memory cells could not be detected in thoracic duct lymph before the 10th day after priming. Thereafter, they increased rapidly in number to reach a plateau by the 4th week. This long latency was not due to a protracted process of maturation of cells formed and delivered to the lymph during the early post-induction period because memory never appeared in rats immunized adoptively with cells obtained before the 11th day. The fact that phiX memory cells are resistant to inhibition by vinblastine also indicates that memory is carried by cells which are released from regionally stimulated lymphoid tissue only after they have ceased dividing and become functionally mature. Adoptively acquired memory decays rapidly at first, but slowly from the 2nd week after cell transfer. This suggests that two cell populations of differing half-life are involved. If two distinct populations are necessary to the full expression of phiX memory, both populations recirculate because the memory cells in lymph draining the sites of the primary response and those reaching the central lymph of adoptive hosts give parallel dose-response curves.


Antibody synthesis in synchronized mouse spleen cells during the secondary response to sheep erythrocytes in vitro. Immunoglobulin synthesis by mouse spleen cells during the secondary response to sheep erythrocytes in vitro was examined in cells synchronized by methotrexate and adenosine treatment. Antibody synthesis by the synchronized cells was discontinuous through the cell cycle and highest in the early S phase. Previous results from this laboratory indicated that antibody secretion, as measured by plaque production to sheep erythrocytes, was also greatest in early S, and taken together these results suggest little difference in the expression of antibody synthesis and secretion through the cell cycle. Actinomycin D was employed in several experiments in an attempt to determine if the variation in antibody synthesis was regulated by transcription of messenger RNA for immunoglobulin. Lower actinomycin concentrations (0.05 mug/ml) reportedly permitting messenger, but not ribosomal, RNA synthesis as well as higher concentrations (1 mug/ml) rapidly reduced immunoglobulin and total protein synthesis. These results indicated that actinomycin was not suitable for determining the stability of messenger RNA in mouse spleen cells. Therefore, the reasons for fluctuation in antibody synthesis during the cell cycle could not be determined, but several possibilities are suggested.