Cystoid macular edema following the implantation of anterior chamber lenses.

Three out of 10 eyes (30%) in which injection molded anterior chamber lenses from McGhan were used developed cystoid macular edema, compared to a much lower incidence with the use of lathe-cut anterior chamber lenses from Rayner.


One man's experience with the Choyce Mark VIII anterior chamber lens.

Twenty implantations of the Choyce Mark VIII lens were accomplished over a one-year period, with an implant moratorium then being placed on this lens by this particular surgeon. I have set forth a tenable explanation for the disheartening results I achieved.


Quantitative studies on the maturation of central and peripheral parts of individual ventral motoneuron axons. II. Internodal length.

The lengths of internodes on peripheral and central stretches of the same ventral motoneuron axons were measured in 6 day rats. The former were more than twice as long as the latter. The distribution of peripheral internodal lengths was broader, and was shifted to the right, relative to that of central internodes. Short internodes were present centrally and peripherally throughout the early period of rapid myelination occurring immediately after birth. All such internodes appeared normal centrally, as did a proportion found peripherally. Some peripheral examples appeared to be degenerating. Evidence is presented suggesting that some short internodes, both peripherally and centrally, increase in length to achieve a normal appearance. Many central stretches of central motoneuron axons which were myelinated at some levels, at other levels possessed unmyelinated segments covered by numberous short glial processes. Such bare stretches commonly occurred immediately deep to the cord surface. The lengths of the unmyelinated stretches decreased as age advanced, perhpas due to the extension of adjacent myelinated segments along them.


A quantitative study of peripheral nerve fibres in the mouse following the administration of drugs. 1. Age changes in untreated CBA mice from 3 to 21 months of age.

This study is concerned with the quantification of changes which occur in peripheral nerves of normal mice from the onset of maturity to old age. The parameters chosen were total fibre area and axon area. Size distributions of these were expressed in the form of histograms. From the area data notional fibre and axon diameters were calculated, together with the thickness of the myelin sheath. The notional diameters were contrasted with the comparable myelin sheath thickness in the form of scatter diagrams. These results are intended to provide a reference scale for subsequent assessment of changes induced by drugs administered throughout life. The are in general accord with previous observations, but were obtained quickly, and with less margins of error, by using a Quantimet 720.


The T-tubule system in the specialized and general myocardium of the rat.

The T-tubule system in cardiac muscle cells has been investigated with the electron microscope in 10 adult rats after infiltration with horseradish peroxidase. All cardiac muscle cells possess a T-system, but its complexity varies according to the region of the heart. It is most extensive in the general ventricular myocardium where there are primary, secondary and tertiary transverse tubules as well as longitudinal elements, and there are numerous couplings between the T-system and the sarcoplasmic reticulum (SR). The T-system and associated SR couplings are less extensive in the atrium and in the atrioventricular conducting system. It is least well developed in the nodes, particularly in the sinuatrial node, where it is restricted to primary T-tubules. There are numerous SR couplings with the sarcolemma in all types of cardiac muscle cells. Where intercalated discs occur, SR couplings are associated with non-specialized parts of the disc. The possible significance of these ultrastructural features in regard to the speed of conduction by myocardial tissues, and in the excitation-contraction sequence is discussed.


Observations on the development of the connective tissues of developing human nerve.

Trochlear nerves from two human fetuses, and digital nerves from a third, have been examined by electron microscopy. Very marked differences in maturation were found between trochlear nerves of fetuses of ages differing only by 2--3 weeks, and between proximal and distal parts of the same trochlear nerve. Immaturity was reflected in paucity of endoneurial space and collagen and in the rarity, or virtual absence, of endoneurial fibroblasts. Circumstantial evidence of collagen formation by Schwann cells has been presented and discussed.


Pyramid-like basket cells in the granular layer of the dentate gyrus in the rat.

Basket cells of the dentate gyrus were identified using Nissl (cresyl violet) staining. It has been found that the ratio between basket and granule cells is 1:150--210. Only a few glial cells, mainly astroglia, were found in the granular layer of the dentate gyrus. In accordance with earlier data it was found that the granule cells and glial cells originate mainly postnatally, but the basket cells, like the pyramidal cells of the hippocampus, originate prenatally.


The effect of position on the migration of muscle.

The tibial insertion of the M. semitendinosus of young rabbits was moved to a new location on the shaft of the tibia, either 5 or 10 mm proximal, or 0, 10, 20, 30, 40, or 50 mm distal to its original position. The animals were killed 8.5 months later. The results show that the greatest amount of proximal migration of the experimental muscles was 11.3 mm and this occurred in the muscles moved 10 mm distally. This compared with a migration of 12.8 mm for control muscles. The amount of proximal migration decreased progressively when the insertion was moved more or less than 10 mm distally. There was a small amount of distal migration in the 10 mm proximal, and the 40 and 50 mm distal groups. These results confirm experiments reported earlier in which muscles were not moved more than 20 mm distally. They support the hypothesis that migration is controlled by the position on the growing bone rather than by the tension in the muscle.


Ultrastructural localization of endogenous peroxidase in human parotid and submandibular glands.

The localization of endogenous peroxidase was studied in human parotid and submandibular glands using the medium of Strum & Karnovsky either at pH 7 or at pH 8.3, after a short fixation of the tissues with a low concentration of glutaraldehyde. At pH 7 reaction product was found in seromucous cells of both glands in the cisternae of the rough endoplasmic reticulum, in the elements of the Golgi apparatus, and in secretory products. However, while in the parotid gland all secretory granules showed peroxidase activity, in the submandibular gland some of the granules were left unstained. At pH 8.3 the pattern of reactivity was practically unchanged in the submandibular gland, whereas the amount of precipitate noticeable in the secretory granules of the parotid gland was greatly reduced. The method of Novikoff et al. for catalase revealed the presence of a number of small bodies having in the characteristics of peroxisomes. Acid phosphatase was not demonstrable in the secretory granules of either parotid or submandibular glands. The lead precipitate indicating enzyme activity was observed only in lysosomes and, occasionally, in the elements of the Golgi apparatus of some sermucous cells.


Neuronal numbers in the superior cervical ganglion of the neonatal rat.

Neuronal numbers in the neonatal rat superior cervical ganglion were estimated from counts made on paraffin wax sections stained with cresyl violet. The raw data were separately processed in accordance with the correction procedures of Abercrombie (1946) and of Hendry (1976). The results do not support the view that there is histogenic neuronal loss during the first 30 days post-partum. Figures corrected by the Abercrombie method indicated that the neuronal population is held stable throughout the first month of neonatal life at a level of about 35000 neurons. On the other hand, when the data was corrected by Hendry's method, the population appears to increase from less than 20000 to about 45000 neurons in the first week, and is then held at that level until the end of the study period. Moreover, histochemical studies using the acid-phosphatase reaction have previously indicated that there is little or no cell degeneration during this period.


Aspects of the human pharyngeal hypophysis in normal and anencephalic fetuses and neonates and their possible significance in the mechanism of its control.

Aspects of the pharyngeal hypophysis in normal and anencephalic human fetuses and neonates have been described. Volumetric and histological changes in the normal gland similar to those observed previously in the adult are noted. The sellar and pharyngeal hypophyses develop in parallel during intrauterine life, but the latter has reached its maximum development by the time of birth. It is suggested that the control of the pharyngeal hypophysis is mediated through factors in the blood, and that the nature of the control and the vascular route vary at particular periods in both fetal and adult life. From a study of the anencephalic material it appears that the individual cells of the pharyngeal hypophysis are capable of marked response to a specific endocrine imbalance, but the capacity of the pharyngeal hypophysis as a whole to compensate significantly for deficiencies of the sellar adenohypophysis is strictly limited by its inability to hypertrophy to any marked degree.


A longitudinal radiographic study of the growth of the rat skull.

The growth of the skull of the rat was longitudinally from 23 to 140 day PC from radiographs. Four measurements were made on each animal at each age studied. The velocity graphs of these measurements showed that for two, namely neurocranium length and bizygomatic width, the peak rate of growth occurred before birth. Viscerocranium length and neurocranium width showed peak rates after birth. Males became significantly larger than females in each of the absolute measurements studied, but in cranial index and facial index there were no significant differences between the sexes. Both these indices showed strain differences when compared to the results obtained by other workers.


The pattern of lymphatic drainage of the rat testis.

The basic pattern of the lymphatic drainage of the testis is described in the mature albino rat following studies in vivo and the examination of dissected specimens in vitro. Microlymphography was undertaken using various contrast media. Three groups of lymphatics leave the surface of the testis--superior, medial and inferior, from the upper pole, from the mediastinum and from the lower pole respectively.


Teaching janitorial skills to the mentally retarded: acquisition, generalization, and maintenance.

A task analysis of janitorial skills required for cleaning a restroom was performed. Six subtasks with a total of 181 component responses were identified. Subjects were required to progress through a series of four prompt levels ordered generally from more to less direct assistance for 20 of the most difficult component steps. Another series of four prompts, ordered from less to more direct assistance, was used to teach the other 161 responses. Subjects progressed to the next more intense prompt level contingent on a failure to respond appropriately with less assistance. A multiple baseline across subjects as well as the six subtasks was employed to evaluate the efficacy of the procedures. Six moderately retarded adolescents were trained in their public school. The results show rapid response acquisition, skill generalization to a second restroom, and maintenance of the newly learned behavior. The present research provides evidence of a model for analyzing and training vocational skills to the mentally retarded.


Measuring client gains from staff-implemented programs.

Confidence in the adequacy with which staff implement training programs requires an analysis of the impact on the client. In two experiments, measures, were devised to reflect this impact. In the first, a measure of the consistency with which clients participated in a toilet-training program revealed their participation to be erratic. Consistent participation occurred after a public display of the consistency of participation was introduced. In Experiment II, detailed measures were devised to reflect the client's performance during the implementation of two physical-therapy programs: range-of-motion and ambulation. Additionally, standardized measures of the benefits that accrued from their participation in these programs were devised. Improvements in both measures were slight and unstable during a condition of immediate feedback (supervisor praise) to staff but substantial improvements were obtained with the addition of a public display of the client's performance.


An index for assessing weight change in children: weight/height ratios.

Weight and height data from two studies were recomputed, and original data were computed using a Weight Index formula that accounts for the interaction of actual weight/height changes in growing children and compares this ratio with that of normed weight/height ratios for equal-aged children. Recomputing the data of one study showed that the Weight Index is a more sensitive procedure for assessing long-term behavioral treatment of obesity for girls than weight alone. Recomputing the data of a second study showed that the Weight Index formula is consistent for boys and girls and is a more sensitive procedure for assessing long-term effects of a variety of medical treatments for obesity than the Ponderal Index or weight alone. Computation of the data for 17 "normal" children in a preschool class showed a zero Weight Index score before and after a six-month interval elapsed without treatment. The procedure may be useful in assessing ponderosity or anorexia over intervals of six months or more with growing children or difference between actual and normed weight over shorter intervals.


Factors influencing mothers' compliance with a medication regimen for asthmatic children.

This study lends support to the utility of a compliance-adapted health belief framework for exploring mothers' differential adherence to medication regiments prescribed for their asthmatic children. Most of the Model components behaved as hypothesized in predicting mothers' drug administration. The findings offer empirical evidence which can be applied to the purposes of identifying potential noncompliers and designating those specific, health-related orientations of the mother which are related to poor cooperation with therapy. Based on an educational diagnosis, the clinician may thus intervene to alter inappropriate health beliefs in order to enhance the likelihood of compliance for the asthmatic patient.


Glucosamine-containing sphingoglycolipids from sheep erythrocytes.

Glucosamine-containing sphingoglycolipids were isolated from sheep erythrocyte membranes, and the presence of glycolipids with long carbohydrate chains was demonstrated. The purification of highly polar glycolipids was achieved by high-performance liquid chromatography of acetylated samples followed by deacetylation with sodium methoxide. Their structures were elucidated by conventional methylation studies, oxidation with chromium trioxide and the direct measurement of permethylated glycolipids by GC-MS. Forssman-active glycolipid2 was a major component of sheep erythrocytes and lacto-N-neotetraosylceramide (LcnOse4Cer) was found to be one of the components. The amount of tetraglycosylceramide was only 5% of that of Forssman-active glycolipid. Three highly polar glycolipid components with ten to twelve carbohydrate residues were also found in sheep erythrocytes.


Cellular factors for stimulation of nucleosomal template activity for in vitro DNA synthesis.

Nucleosomes isolated from Yoshida sarcoma chromatin by micrococcal nuclease treatment were relatively inactive as templates for in vitro DNA synthesis. However, the template activity increased by trypsin digestion of nucleosomes or addition of heparin to the reaction mixture. This indicates that the nucleosomal template activity is masked. A crude extract of Yoshida sarcoma cells stimulated the nucleosomal template activity. The stimulatory factor was separated into three peaks by DEAE cellulose column chromatography. The same three peaks were observed in normal rat liver extract with much lower activities, but enhanced in regenerating liver. The factors seem to stimulate DNA synthesis by activating DNA template in nucleosomes without degrading histones or changing the primary structure of nucleosomal DNA.


Methylamine dehydrogenases of Pseudomonas sp. J and Pseudomonas AM1. Study on subunit structure by dimethyl suberimidate.

Methylamine dehydrogenase (MW: 105,000) of Pseudomonas sp. J was treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked proteins having different molecular -eights of 53,000, 64,000, 80,000, 93,000, and 103,000 were found in addition to 13,000 (light subunit) and 40,000 (heavy subunit) by SDS polyacrylamide gel electrophoresis. Isolated light and heavy subunits were separately treated with the reagent. The product having a molecular weight of 80,000 was found to be a major cross-linked protein for the heavy subunit but no product was found for the light subunit. A similar electrophoretic pattern was also obtained for the reconstituted enzyme from the subunits of Pseudomonas sp. J and for methylamine dehydrogenase of Pseudomonas AM1. These results suggest that methylamine dehydrogenases obtained from these two bacteria are both the alpha2beta2-type subunit enzyme and have a geometrically analogous subunit structure.


Selective cleavage of peptide bonds by a serine protease from rat skeletal muscle.

The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-Glu, were not susceptible to another serine protease from small intestine (SI-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the SI-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the SI-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the SI-protease.


Metabolism of putrescine to 2-pyrrolidone by rat liver slices.

The metabolism of [14C]putrescine was studied in sliced rat liver in vitro. delta1-Pyrroline and gamma-aminobutyric acid, known metabolic products of putrescine, were found in the medium. However, 2-pyrrolidone was the major product of the metabolism of putrescine secreted into the medium. 2-Pyrrolidone was also synthesized from putrescine by spleen and lung, but not by kidney, brain, heart, or muscle. This is the first demonstration of the synthesis of 2-pyrrolidone in a biological system.


Studies on cobalt myoglobins and hemoglobins. Proton magnetic resonance investigation of the subunit interaction in iron-cobalt hybrid hemoglobins.

The paramagnetically shiftedd proton nuclear magnetic resonance spectra of iron-cobalt hybrid hemoglobins [alpha(Co)2beta(Fe)2 and alpha(Fe)2beta(Co)2], as well as those of deoxy forms of cobalt hemoglobin, iron hemoglobin, and their isolated chains, have been measured at 360 MHz. The proton NMR signals of the deoxy forms of iron and cobalt hemoglobins were individually assigned to each subunit. The NMR spectral characteristics of the alpha subunits in deoxycobalt hemoglobin, as well as those in deoxy-alpha(Co)2beta(Fe)2, were found to be quite different from those of beta(Co)2 subunits or isolated alpha-SH chain. Upon ligation of carbon monoxide to the beta(Fe)2 subunits in alpha(Co)2beta(Fe)2, the spectral properties of deoxy-alpha(Co)2 subunits became similar to those of the deoxy-beta(Co)2 subunits. No significant change in the NMR spectrum of the beta(Co)2 subunits was observed in alpha(Fe)2beta(Co)2 upon ligation of carbon monoxide to the alpha(Fe)2 subunits. These observations show the linkage of the electronic structure of the prosthetic groups with the subunits cooperativity in hemoglobin, as well as the inequivalence of the subunits. This is the first report on the paramagnetically shifte proton NMR spectra of the cobalt-substituted hemoproteins.


Structural evidence for methionine at the reactive site of human alpha-1-proteinase inhibitor.

An unadecapeptide, obtained by papain digestion of denatured human alpha-1-proteinase inhibitor (alpha-1-PI), has been isolated and sequenced. The structure of this fragment overlaps with the NH2-terminal sequence of modified inhibitor (alpha-1-PI) prepared from dissociated complexes of alpha-1-PI with trypsin, chymotrypsin, and elastase. Furthermore, structural homology with the reactive centers of proteinase inhibitors from other sources is readily detectable. Methionine has been found to occupy the apparent P1 position in alpha-1-PI and the potential inactivation of the inhibitor by oxidation of this critical residue may be important in obtaining a biochemical link with the development of lung disease.


Membrane potential-sensitive fluorescence changes during Na+-dependent D-glucose transport in renal brush border membrane vesicles.

When D-glucose was added to a suspension of renal brush border membrane vesicles equilibrated in a Na+-containing medium, there was a rapid transient increase in the fluorescence of the probe, 3,3'-dipropyl thiodicarbocyanine iodide (DiS-C3-(5)). This sugar-induced response was stereospecific for the D isomer, dependent on Na+, inhibited by phlorizin, and blocked by ionophores, valinomycin plus nigericin, which dissipate ionic gradients. The enhancement in fluorescence suggests the entrance into the vesicle of Na+, cotransported with the sugar. This would lead to the interior of the membrane vesicle becoming more positive, resulting in depolarization of the membrane potential. That the sugar induced the transport of Na+ was confirmed by direct measurement of 22Na+ uptake. Thus, the Na+-sugar co-transport system provides a mechanism for D-glucose to stimulate the flux of Na+ as well as for the Na+ electrochemical gradient to enhance the transport of D-glucose.


Preparation of protease-free and ribonuclease-free pancreatic deoxyribonuclease.

When pancreatic DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, freedom from contaminating ribonucleases or proteases is an important property of the enzyme preparation. A simple one-step procedure has been developed to effect complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose (a column (0.9 X 60 cm) operated in series with a regeneratable 1-ml bed). Commercial preparations of DNase (about 10 mg) give a quantitative yield of the enzyme that is protease-free as evidenced by full stability for more than 10 days at pH 8 and 37 degrees C even in the absence of the protecting action of Ca2+. Removal of the last traces of RNase has been accomplished by affinity chromatography on a column (0.4 X 72 cm) of 5'-(4-aminophenyl-phosphoryl)-uridine 2'(3')-phosphate-Sepharose; the product is a highly active DNase that gives no detectable hydrolysis of RNA by assay on radioactive substrates.


Farnesyl pyrophosphate synthetase. Mechanistic studies of the 1'-4 coupling reaction with 2-fluorogeranyl pyrophosphate.

The mechanism of the 1'-4 coupling reaction between isopentenyl pyrophosphate and geranyl pyrophosphate catalyzed by farnesyl pyrophosphate synthetase from porcine liver was studied with the allylic substrate analogue 2-fluorogeranyl pyrophosphate. 2-Fluorogeranyl pyrophosphate is an alternate substrate for the enzyme, yielding 6-fluorofarnesyl pyrophosphate upon condensation with isopentenyl pyrophosphate. The Michaelis constant for the fluoroanalogue, Km = 1.1 micron, is similar to that measured for geranyl pyrophosphate, Km = 0.7 micron. However, the rate of condensation with the fluoroanalogue was only 8.4 X 10(-4) that of the normal reaction. A similar rate of depression (4.4 X 10(-3)) was found for solvolysis of geranyl methanesulfonate and the corresponding 2-fluoro derivative, reactions known to proceed via cationic intermediates. In contrast, displacement of chlorine from geranyl chloride and 2-fluorogeranyl chloride by cyanide showed a small (2-fold) rate enhancement for the fluoro compound. Finally, 2-fluorogeranyl pyrophosphate is a competitive inhibitor against geranyl pyrophosphate. These data are interpreted in terms of an ionization-condensation-elimination mechanism for the 1'-4 coupling reaction.


Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions.

The time course of methemoglobin reduction by ascorbic acid under anaerobic conditions was analyzed by using isoelectric focusing on Ampholine plate gel in order to compare results obtained by studies of the changes in absorption during the reaction. The intermediate hemoglobin which appeared all through the reaction was single and identified as the alpha3+beta2+ valency hybrid. In the presence of inositol hexaphosphate, reduction of methemoglobin was considerably accelerated and this acceleration was restricted to the step in which methemoglobin is reduced to the alpha3+beta2+ valency hybrid. The phase containing alpha3+beta2+ valency hybrid reduction to deoxyhemoglobin was not affected by the presence of this organic phosphate. The reaction rate constant of each phase was estimated by the analysis of the changes in three components such as methemoglobin, alpha3+beta2+ valency hybrid, and deoxyhemoglobin. The specific attack of beta-methemoglobin chains in methemoglobin tetramer by ascorbic acid was explained by the functional differences in alpha- and beta-methemoglobin chains in the protein.


Orientation of the band 3 polypeptide from human erythrocyte membranes. Identification of NH2-terminal sequence and site of carbohydrate attachment.

The NH2-terminal sequence and carbohydrate attachment site of the 95,000-dalton transmembrane polypeptide (Band 3) from human erythrocyte membranes have been studied. The blocked NH2-terminal sequence is Ac-Met-Glu-Glu; the presence of this sequence in specific fragments of the polypeptide confirms that the end of the polypeptide which is inside the cell is the NH2-terminal. The carbohydrate associated with the Band 3 polypeptide appears to be attached at a single site in the COOH-terminal third of the molecule, to a region with composition Asx1Ser2; this confirms that part of the polypeptide toward the COOH-terminal is outside the cell. The carbohydrate structure appears to be extremely heterogeneous both in size and composition, which probably causes the Band 3 polypeptide to migrate as a diffuse band on dodecyl sulfate gel electrophoresis.


Kinetic analysis of rabbit liver tRNA nucleotidyltransferase.

The kinetic properties of rabbit liver tRNA nucleotidyltransferase were examined. Our results indicate that incorporation of AMP into tRNA-C-C at pH 9.4 proceeds by a rapid equilibrium Random Bi Bi mechanism. This conclusion was supported by bisubstrate initial velocity studies, dead end and product inhibition, and isotope exchange at equilibrium and during the net reaction. This analysis has made possible calculation of the dissociation constants of various substrates and substrate analogs. The data indicate that binding of either a substrate or substrate analog greatly decreases the affinity of the enzyme for the second substrate. Comparison of binding constants for tRNAs revealed that affinities for the enzyme are tRNA-C-C greater than tRNA-C-Cp greater than tRNA-C-C-A. Also, ATP binds considerably more tightly than ADP. The apparent equilibrium constant of the forward reaction was determined and found to be about 1500 at pH 9.4, and about 50 at pH 7.0. Analysis of the reaction at pH 7.0 suggested that it proceeds by a similar mechanism, but that the dissociation of tRNA-C-C-A becomes more important. The relation of these in vitro properties of the enzyme to its cellular function are discussed.


Glucose transport characteristics of quiescent thymocytes.

Rat thymocyte populations contain "active" cells which equilibrate quickly with 3-O-methylglucose and "quiescent" cells which equilibrate slowly. Glucose transport stimuli make quiescent cells behave like active cells as regards glucose transport. Glucose transport in quiescent cells was inhibited by phloretin and cytochalasin B. Active cells were heterogeneous in their susceptibility to inhibition by 0.1 micron cytochalasin B, half of them being inhibited about 95%, while half were inhibited modestly. The methylglucose entry Km of quiescent cells was 3 times that of active and stimulated cells. The methylglucose entry Vmax of quiescent cells was one-eighth that of stimulated cells. A fixed internal methylglucose concentration increased the influx Km of quiescent cells but not that of active or stimulated cells. These findings and others are compatible with a model in which the carrier of quiescent cells is masked by an alteration (such as ligand binding) which is relatively specific for empty as opposed to sugar-loaded carrier and rather indifferent as to in versus out carrier orientation. The masking alteration stabilizes inward-facing and outward-facing unloaded carrier against both translocation and loading. The data fail to show evidence of a masking alteration of loaded carrier.


Structures of the acidic oligosaccharides isolated from rat sublingual glycoprotein.

Alkaline borohydride reductive cleavage of the major glycoprotein fraction of rat sublingual gland resulted in the release of a series of reduced oligosaccharides. Five sialic acid-containing oligosaccharides composed of 9, 10, 12, 13, and 15 sugar residues, respectively, have been purified from the acidic oligosaccharide fraction. The structures of these oligosaccharides, as determined by sequential digestion with specific glycosidases, periodate oxidation and methylation analyses of the native and desialyzed compounds, are proposed to be (formula: see text).


Association of diphtheria toxin with Vero cells. Demonstration of a receptor.

The interaction of radiolabeled diphtheria toxin with highly sensitive mammalian cell lines was studied. Toxin bound to (or was taken up by) Vero cells at 4 and 37 degrees C in a highly specific manner. At both temperatures, excess unlabeled toxin competed for up to 90% of the cell-associated label. The association at 37 degrees C was biphasic, increasing to a peak at 1 to 2 h and falling thereafter. At 4 degrees C, association increased with time to a steady state. Both fragment B and CRM-197 competed for the association of labeled toxin with cells. The magnitude of association correlated with the cytotoxic sensitivity of several cell lines. Both pH and exogenous nucleotides affected the association in a manner consistent with effects on cytotoxicity. The label associated with cells at 4 degrees C was largely intact toxin, while that at 37 degrees C was degraded. At 4 degrees C, the association was saturable (K = 9 X 10(8) liters/mol), was reversible, and indicated about 1 to 2 X 10(5) binding sites/cell.


Carbon monoxide binding to a fish hemoglobin under photostationary conditions.

Determinations of carbon monoxide binding curves for hemoglobin from Brevoortia tyrannus under equilibrium and photostationary conditions show that in the light, the curve is shifted to the right and altered in shape. The Bohr effect is much less in the light. The kinetics of the transition between equilibrium and photostationary states has been examined. All of the results are satisfactorily described using the two-state model of Monod, J. Wyman, J., and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118 with the assumption that light produces an additive increase in the rate of dissociation of ligand from the R and T states.


Isolation of monomeric cytochrome f from Japanese radish and a mechanism of autoreduction.

Monomeric cytochrome f from Japanese radish (Raphanus sativus L. var acanthiformis Makino) leaves was isolated in a homogeneous state with an A420.5/A277 of 7.6. Radish cytochrome f is a single polypeptide chain with a molecular weight of about 33,000. The midpoint potential is 350 mV. The amino acid analysis indicates the existence of 3 residues of half-cystine. Radish cytochrome f contains one thiol group which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) only after denaturation by sodium dodecyl sulfate. Ferricytochrome f is reduced by the superoxide radical at the rate of 6 X 10(6) M-1 s-1 at pH 7.8. Radish ferricytochrome f is also reduced slowly without an exogenous electron donor. A kinetic study and the effect of the thiol reagent indicate that the autoreduction is an intramolecular reaction and that the thiol group is an electron donor.


The asparagine-linked sugar chains of subcomponent C1q of the first component of human complement.

Human C1q, a subcomponent of the first component of complement, contains six asparagine-linked sugar chains in 1 molecule. The sugar chains are exclusively located in the COOH-terminal globular region which is composed of 330 amino acid residues. The sugar chains were liberated from the polypeptide portion by hydrazinolysis, and their structures were studied by the combination of sequential exoglycosidase digestion and methylation analysis. Based on the results, the structures NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6(+/- NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4(+/-Fucalpha1 leads to 6)GlcNAc were confirmed.


Characterization of intermediate hemoglobin produced during methemoglobin reduction by ascorbic acid.

Methemoglobin reduction by ascorbic acid was found apparently to cease halfway without further reduction. Studies by isoelectric focusing on Ampholine plate gel revealed that the solutions of the halfway reduced methemoglobin are composed of about 6% oxyhemoglobin, 59% intermediate hemoglobin, and 35% methemoglobin. The intermediate hemoglobin was isolated by CM Sephadex C-50 column chromatography and identified as alpha3+beta2+ valency hybrid by studies using the pattern of isoelectric focusing of p-chloromercuribenzoate-treated intermediate hemoglobin on Ampholine plate gel, absorption spectra, and difference spectra induced by the addition of inositol hexaphosphate in comparison with the reconstituted valency hybrids, alpha3+beta2+ and alpha2+beta3+. Essentially no alpha2+beta3+ valency hybrid was included in the intermediate hemoglobin solutions. These results suggest that methemoglobin reduction by ascorbic acid is mainly initiated by the attack of beta-methemoglobin chains accompanied by the following scheme. Methemoglobin leads to alpha3+beta2+ valency hybrid leads to oxyhemoglobin. The course of methemoglobin reduction by ascorbic acid through alpha2+beta3+ is likely to be small.


Enzyme-activated irreversible inhibitors of L-ornithine:2-oxoacid aminotransferase. Demonstration of mechanistic features of the inhibition of ornithine aminotransferase by 4-aminohex-5-ynoic acid and gabaculine and correlation with in vivo activity.

L-Ornithine:2-oxoacid aminotransferase is a specific enzyme with respect to the amino group donor. Nevertheless it was found that this enzyme is inhibited by some 4-aminobutyrate analogs, 4-aminohex-5-ynoic acid and 5-amino-1,3-cyclohexadienyl-carboxylic acid (gabaculine), which are currently considered to be enzyme-activated irreversible inhibitors of 4-aminobutyrate:2-oxoglutarate aminotransferase. The inhibitory mechanisms for the two omega-aminotransferases are identical. A close structural analog of these inhibitors, 4-aminohex-5-enoic acid, is not inhibitory for ornithine aminotransferase, whereas it effectively inhibits 4-aminobutyrate aminotransferase. The reasons for this difference are discussed. The in vitro findings are entirely transferable to the in vivo situation: 4-aminohex-5-ynoic acid and gabaculine cause a long-lasting inhibition of ornithine aminotransferase in brain and liver, and reduce significantly in vivo ornithine degradation, whereas 4-aminohex-5-enoic acid is inactive both in vivo and in vitro toward this enzyme. The enzyme-activated irreversible inhibitors allow one for the first time to study the physiological consequences of irreversible ornithine aminotransferase inhibition.


Comparison of the NH2-terminal sequence of ovalbumin as synthesized in vitro and in vivo.

The sequence of the NH2-terminal 35 residues of chicken ovalbumin was found to be identical with that of the product translated in vitro from the corresponding mRNA. Together with our previous results (Palmiter, R.D., Gagnon, J., and Walsh, K.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 94-98), these data show that the only co-translational processing event at the NH2 terminus of the primary translation product is the replacement of the initiator methionine by an acetyl group. Thus, secretion of this major protein by chick oviduct does not involve the cleavage of a "signal peptide" as is characteristic of three other egg white proteins secreted by the same cells.


Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides.

Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.


Fractionation of human parotid saliva proteins.

A chromatographic procedure for purification of the proteins in human parotid saliva has been developed. The eluates of a Sephadex G-150 and two ion exchange columns have been analyzed simultaneously by several physical and chemical tests; these include three optical properties of proteins, assays for neutral sugars, sialic acid and zinc, and disc gel electrophoresis. The ratios of the different variables have been used to determine the homogeneity and complexity of the protein distribution in the various peaks of the chromatographic eluates. By chromatographic methods, it has been possible to purify a glycoprotein with unusual staining characteristics and amino acid composition. Glycoproteins with similar properties comprise a major portion of the proteins in parotid saliva and appear to constitute a family of related proteins which differ in molecular size, carbohydrate and sialic acid content, and electrophoretic mobility. The fractionation of several enzymes in parotid saliva is also reported.


Development of hormone receptors and hormone responsiveness in vitro. Effect of prolonged insulin treatment on hexose uptake in 3T3-L1 adipocytes.

Exposure of insulin-responsive, differentiated 3T3-L1 cells (adipocytes) to 0.1 to 1.0 microgram/ml of insulin for 3 to 48 h resulted in a persistent state of enhanced 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Elevated basal transport activity was retained under conditions where 125I-insulin binding activity remained unchanged and exchangeable insulin was dissociated from cell surface receptors. The appearance of enhanced hexose transport activity was prevented by cycloheximide and could be distinguished from the activation of the glucose transport system observed in these and other cells during glucose deprivation. Rigorously washed adipocytes, exhibiting insulin-induced elevations in basal transport activity, were refractory to further stimulation by insulin during hexose uptake assays. Insulin-unresponsive 3T3-L1 preadipocytes failed to increase hexose uptake activity when  treated under conditions that elicited an optimal response in adipocytes.


Subunit composition and molecular weights of the developmentally regulated lectins from Dictyostelium discoideum.

Discoidin I, the developmentally regulated carbohydrate binding protein from Dictyostelium discoideum, migrated as a broad band during electrophoretic analysis using 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The band could be resolved into two components by electrophoresis on 15% polyacrylamide gels. All highly purified preparations of discoidin I contained equal amounts of the two components, suggesting they are nonidentical subunits of the protein. The subunit molecular weights of discoidin I and discoidin II were found to be 29,500 and 26,500, values somewhat larger than those previously reported.


Hemin controls the expression of the beta minor globin gene in Friend erythroleukemic cells at the pretranslational level.

When clone 745 Friend erythroleukemia cells are induced to differentiate by treatment with 1 X 10(-4) M hemin, the beta minor globin gene is preferentially expressed over the beta major gene. An analysis of the beta-mRNA molecules in in vitro translation systems indicates that essentially only the beta minor message is available for translation. This indicates that in Friend erythroleukemia cells hemin selectively controls the expression of the beta minor globin gene at the pretranslational level.


The different molecular weight forms of bovine liver rhodanese.

Sedimentation equilibrium studies show that there are two forms of bovine liver rhodanese in crystalline enzyme preparations of full specific activity. One form dissociates to a species with a limiting molecular weight close to 19,000. The second form is nondissociable under the same experimental conditions. It exhibits a molecular weight of approximately 33,000. These conclusions are augmented by data from polyacrylamide gel electrophoresis and molecular exclusion chromatography. They serve to explain apparent inconsistencies in previous reports on this enzyme.


Peroxisomal localization of serine:pyruvate aminotransferase in human liver.

The distribution of L-serine:pyruvate aminotransferase (EC 2.6.1.51) in human liver was examined by centrifugation in a sucrose density gradient. The enzyme was located only in the peroxisomes and in the soluble fraction. The peroxisomal and soluble enzymes were highly purified and characterized. The two enzyme preparations had nearly identical properties, suggesting that the soluble enzyme is from broken peroxisomes. The two enzyme preparations showed different properties from rat liver serine:pyruvate aminotransferase (Noguchi, T., Okuno, E., and Kido, R. (1976) Biochem. J. 159, 607-613).


Postsynthetic modification of high mobility group proteins. Evidence that high mobility group proteins are acetylated.

High mobility group proteins were isolated from calf thymus and duck erythrocyte nuclei and the possibility was investigated that these proteins undergo acetylation similar to that occurring in some histones. Dinitrophenylation of the proteins followed by acid hydrolysis and amino acid analysis indicated that 2 to 3% of the lysine residues present were unavailable for reaction with fluorodinitrobenzene. Extensive enzymatic degradation with trypsin and pronase and subsequent amino acid analysis showed a significant amount of material eluting at the position of epsilon-N-acetyllysine. Recovery and acid hydrolysis of this material generated a peak eluting in the lysine position. In vitro radioactive labeling of calf thymus nuclei with [3H]acetate yielded labeled high mobility group proteins. All of these findings are in accord with the conclusion that high mobility group proteins are acetylated and that acetylation occurs as a postsynthetic modification of these proteins.


Glycosylation of interferons. Effects of tunicamycin on human immune interferon.

Human immune interferon, induced in leukocytes by phytohemagglutinin, was prepared in the absence and presence of tunicamycin, an antibiotic which selectively inhibits the glycosylation of newly synthesized glycoproteins. Interferon preparations, produced in the absence of the antibiotic, displayed a considerable chromatographic heterogeneity on: (a) concanavalin A-agarose, (b) phenyl-agarose, (c) Cibacron Blue F3GA-agarose, and (d) polyuridylic acid-agarose. This heterogeneity was completely eliminated when tunicamycin (2 microgram/ml) was present during induction of interferon; all activity was then recovered in the breakthrough fractions from all sorbents. The level of interferon activity in leukocyte culture fluid was not affected by tunicamycin within the range of concentration 0.05 to 2.0 microgram/ml. These data indicate that (a) human immune interferon undergoes glycosylation, and tunicamycin is an effective inhibitor of this process. Thus, it appears that (b) at least some of the carbohydrates of human immune interferon are N-glycosidically linked. Moreover, it seems that (c) glycosylation is not necessary for an interferon molecule to either be secreted by the cell or (d) to express its antiviral function. Such properties of human immune interferon as (e) the apparent hydrophobicity and (f) an affinity for a polyribonucleotide are conferred only when its glycosylation is unimpaired.


Electron-accepting properties of cytochrome o purified from Vitreoscilla.

The reduction of cytochrome o which contains two hemes/molecule required two electrons/molecule when titrated with dithionite under anaerobic conditions. Two types of spectral transitions occurred during this reduction, first a decrease in absorption bands for the oxidized protein at 540 and 405 nm and then a shift in the absorption maxima to 555 and 425 nm, respectively. Each of these two transitions required approximately one electron, evidence that the two hemes reduced separately. Preliminary estimates of the midpoint potentials of the two hemes enabled the selection of two suitable oxidation-reduction dyes, toluylene blue (E'o = +0.115 V) and indigo carmine (E'o = -0.125 V) which were used to estimate more accurately the midpoint potentials of the high and low potential hemes, respectively, using equilibrium photochemical titrations with EDTA and flavin mononucleotide. The midpoint potentials of the two hemes of cytochrome o determined by this technique were +0.118 and -0.122 V.


Copolymers of chondroitin 4-sulfate and chondroitin 6-sulfate in chick embryo epiphyses and other cartilage.

Tetrasaccharides were prepared from chondroitin sulfate by means of a limited degradation with chondroitinase ABC. Tetrasaccharides containing one sulfate per disaccharide unit were isolated and were found to be of three types: a tetrasaccharide with two 6-sulfated disaccharide units, a tetrasaccharide with two 4-sulfated disaccharide units, and a tetrasaccharide with one 4-sulfated disaccharide unit and one 6-sulfated disaccharide unit. Samples of each of these three types of tetrasaccharides were obtained from chick embryo epiphyseal cartilage and from a mixture of bovine tracheal cartilage and shark cartilage. The presence of both a 4-sulfated disaccharide unit and a 6-sulfated disaccharide unit in the same tetrasaccharide molecule indicates the existence of mixed 4 and 6 sulfation on the same chondroitin sulfate chain.


mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Purification and physical properties.

An S-adenosyl-L-methionine:mRNA(nucleoside-2'-)-methyltransferase, one of at least three activities required for the 5'-terminal modification of mRNA, has been purified from vaccinia virus particles. Employing brome mosaic virus RNA ending in m7G(5')pppG- as substrate, a simple DEAE-cellulose filter assay measuring the incorporation of methyl groups from S-adenosyl[methyl-3H]methionine to position 2' of the penultimate nucleoside was devised. Starting from disrupted vaccinia virus cores, a 350-fold enzyme purification was achieved by successive chromatography on columns of DEAE-cellulose, CM-Sephadex, and APP-agarose. Analysis of the isolated enzyme by sodium dodecyl sulfate-polyacrylamide discontinuous gel electrophoresis revealed a single polypeptide with a molecular weight of 38,000. Similar molecular weights were obtained by sucrose gradient centrifugation and gel filtration of the native methyltransferase. The isoelectric point of the purified enzyme occurs at pH 8.4.


mRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Characteristics and substrate specificity.

An mRNA(nucleoside-2'-)-methyltransferase purified from vaccinia virus was shown to methylate the penultimate nucleoside of RNA ending in m7G(5')pppN-. By contrast, RNAs ending in pN-, ppN-, or even G(5')pppN- are not methyl acceptors. This specificity indicates that 2'-O-methylation is the final step in the formation of the m7G(5')pppNm- cap structure. Both adenosine and guanosine are methylated, in accordance with the presence of these nucleosides in the penultimate position of vaccinia virus mRNAs. Studies with homopolyribonucleotides containing m7G(5')pppN ends indicated that poly(A) and poly(I) were the best methyl acceptors while significant but much less activity was obtained with poly(G), poly(U), and poly(C). Simple dinucleotides of the type m7G(5')pppN, however, are poor substrates and do not compete with capped RNA. Additional studies indicate that the methyltransferase has a pH optimum of 7.5, does not require divalent cations, is inhibited by S-adenosylhomocysteine, has a Km of 2.0 micrometer for S-adenosylmethionine, a Km of approximately 5 nM for brome mosaic virus RNA, and kinetics consistent with a random bireactant mechanism.


Release of phospholipase A and triglyceride lipase from rat liver.

A new, rapid, and sensitive assay for phospholipase A, utilizing commercially available [14C]phosphatidylethanolamine with 14C label in both palmitic acid moieties, was used to study phospholipase A release from perfused liver, hepatocytes, and intestinal cells from rats. Heparin triggered a prompt release of phospholipase A from perfused liver. Phospholipase A and triglyceride lipase were released from hepatocytes at a linear rate for 1 h and 30 min, respectively. Heparin (20 u/ml) doubled the release of phospholipase A and triglyceride lipase from hepatocytes. Colchicine (0.1 mM), but not puromycin (0.2 mM), inhibited basal and heparin-stimulated phospholipase A release by 40%. Since the amount of phospholipase A and triglyceride lipase released into the medium greatly exceeded intracellular activities, it is possible that secretion is coupled with intracellular conversion from inactive to active forms of the enzymes. Dibutyryl cyclic AMP (1 mM) inhibited phospholipase A (48%) and triglyceride lipase (82%) release from hepatocytes. Epinephrine, dexamethasone, and clofibrate inhibited release of triglyceride lipase but not phospholipase A. Phospholipase A activity of intestinal cells was greater than in hepatocytes, but neither heparin nor dibutyryl cyclic AMP affected phospholipase A release from intestinal cells. These results suggest that the liver is a major source of phospholipase A of postheparin plasma. The fact that dibutyryl cyclic AMP affects the release of these enzymes suggests an additional mechanism for hormonal regulation of lipid and lipoprotein metabolism.


De novo purine synthesis in avian liver. Co-purification of the enzymes and properties of the pathway.

The enzymes of the de novo purine biosynthetic pathway have been partially co-purified from pigeon liver by a method dependent upon the use of the nonionic polymer polyethylene glycol for enzyme stabilization and cofractionation. Although the enzymes did not appear to constitute a large macromolecular complex it was evident that some particular inter-relationship between them was preserved during the purification procedure. Analysis of the end products and pathway intermediates was carried out primarily by sensitive high pressure liquid chromatographic techniques. Substrate and cofactor requirements were confirmed and optimal conditions of pH, temperature, and K+ ion activation established. At phosphoribosyl pyrophosphate (PP-ribose-P) concentrations below 0.3 mM the activity of the first pathway enzyme amidophosphoribosyltransferase was rate-limiting, and the inhibition of this enzyme by AMP regulated the rate of purine ring synthesis. At higher concentrations of PP-ribose-P, aminoimidazole ribonucleotide synthetase, the fifth enzyme of the pathway became rate limiting and was subject to inhibition by added AMP. It was evident that the regulation of purine synthesis was quite complex and that AMP inhibition (perhaps reflected in a low adenylate energy charge) can be effected at different points on the purine pathway.


Assignment of the cysteinyl 13C nuclear magnetic resonances and comparison of other aliphatic amino acid resonances of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins.

13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin. Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms. The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to [4Fe-4S] clusters of the oxidized and reduced ferredoxins. The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding [4Fe-4S] clusters in each protein are similar.


Selective binding of mouse estradiol.receptor complexes to oligo(dT)-cellulose.

As a model for the nonspecific interaction of steroid receptors with DNA, the binding of estradiol.receptor complexes of mouse kidney and uterine cytosols to oligo(dT)-cellulose was studied in detail. A limited concentration range of monovalent cationic salts was required for optimal binding, regardless of prior activation of the receptor complexes or the oligomer ligand. Thermal activation of the receptor complexes did not facilitate binding. The reaction was selective for intracellular steroid hormone.receptor complexes, as extracellular proteins binding estradiol with low affinity (bovine serum albumin) or high affinity (mouse alpha-fetoprotein) were inactive. Both crude and partially purified kidney cytosol receptor complexes bound preferentially to oligo(dT)- and oligo(dC)-celluloses, rather than oligo(dA)-celluloses. These findings suggest that at least part of the nonspecific interaction of estradiol.receptor complexes with native DNA is through a salt-sensitive binding of the complex to pyrimidine-rich surfaces of the DNA.


Mechanism of luteinizing hormone regulation of prostaglandin synthesis in rat granulosa cells.

The present study was undertaken to examine the mechanism by which luteinizing hormone (LH) stimulates prostaglandin (PG) synthesis in rat granulosa cells. Immature rats were injected with 20 IU of pregnant mare's serum gonadotropin and granulosa cells isolated 48 h later. LH (5 microgram/ml) stimulated PGE synthesis markedly over the control with no additions in 6-h incubations (1.98 +/- 0.24 and 0.24 +/- 0.05 ng/2 X 10(6) cells, respectively, n = 6). When arachidonic acid (100 microgram/ml) was included during the last hour of incubation, further increases to 15.4 +/- 2.9 and 2.48 +/- 0.48 ng of PGE/2 X 10(6) cells in LH and control incubations were observed. The cause of the increased response to a 1-h incubation with arachidonic acid in the LH-treated cells did not appear to be a stimulation of fatty acid uptake. In addition, when cellular lipids were labeled by a 2-h incubation with radioactive arachidonic acid, LH did not stimulate intra- or extracellular release of arachidonic acid. A 5-fold stimulation of prostaglandin synthetase activity, however, was observed in cells incubated with LH for 5 h. Our results, therefore, indicate that LH acts at a step in the prostaglandin pathway after hydrolysis of arachidonic acid esters and produces an increase in prostaglandin synthetase activity.


Inhibition of elongation steps of protein synthesis at reduced potassium concentrations in reticulocytes and reticulocyte lysate.

We treated intact reticulocytes with nystatin to make them permeable to small cations and varied the concentration of K+ in the medium; the ionic strength was kept constant by adjusting Na+. As the intracellular K+ was lowered, the rate of protein synthesis decreased. In a reticulocyte lysate, the dependence of the rate of protein synthesis on K+ concentration (at constant ionic strength) was similar to that seen in nystatin-treated cells. We observed no loss of polysomes at low K+. Even when initiation was blocked with aurintricarboxylic acid, decreased K+ inhibited protein synthesis. Analysis of the kinetics of synthesis showed that lowering the K+ concentration inhibits the rate of elongation of nascent chains, but has little effect on the relative rate of initiation of chains.


Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate.

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increased and MgCl2 lowered slightly the protective effect of ribulose-1,5-bisphosphate. Modification of sulfhydryl groups by p-chloromercuribenzoic acid inhibited the enzyme and excluded binding of 8 equivalents of pyridoxal 5'-phosphate. Upon removal of the mercuribenzoate groups with excess dithiothreitol, the loss of enzyme activity was exponentially correlated with the binding of 8 mol of pyridoxal 5'-phosphate/mol of enzyme. In contrast to p-chloromercuribenzoic acid, iodoacetamide, which inhibited the catalysis, had no influence on the binding of the 16 pyridoxal 5'-phosphate/mol of enzyme. It is suggested that the CO2 activator site with one epsilon-amino group for binding of CO2 and the catalytic site with one epsilon-amino group of a yet unknown function are located closely together on the large subunits of the enzyme. The results are consistent with a sulfhydryl group as the proton acceptor opposite carbon 3 of ribulose-1,5-bisphosphate, and when this sulfhydryl group is blocked by the bulky mercuribenzoate group, but not by the small carboxyamidomethyl group, pyridoxal 5'-phosphate binding is modified.


Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.


Isolation and characterization of flavin-linked glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria and comparison with the enzyme from rabbit brain.

Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.


Enzyme immobilization on heparin.

We describe the preparation and some of the properties of heparin-bound alpha-chymotrypsin that were obtained via activation of heparin with water-soluble carbodiimide. Immobilized enzyme has unchanged kinetic characteristics toward low-molecular- weight and macromolecular substrates. The heparin-bound enzymes could have a wide range of medical applications.


Transport properties of hydrolyzed polyacrylonitrile.

Measurements of the permeability coefficients of various compounds up-to a molecular weight of about 70,000 have shown clearly that membranes prepared from hydrolyzed polyacrylonitrile are about 10 times more permeable than those made of poly(2-hydroxyethyl) methacrylate. The higher permeability is probably due more to the higher water content (about 75%) than to the type of network. The high mechanical strength of the membranes and their good permeability to compounds possessing a comparatively high molecular weight seem to designate the material for some new applications in medicine.


Interactions of bovine thrombin and plasma albumin with low-energy surfaces.

Surface configurations are vessels fabricated from tubing and plate, films deposited on the surface of vessels, and beads confined in vessels. The average association constant between thrombin and sites on commercial poly(methyl methacrylate) surface (Lucite) is near 4 X 10(8) liters/mole at 22 degrees C, pH 7.0, and ionic strength 0.15. Depending on Lucite composition, average adsorption U, in molecules/cm2 of apparent solution-surface interface, ranges from 0.7 to 8.8 X 10(11). Analysis based on the assumptions that solution dimensions are preserved, adsorption is random, and surface rearrangement is negligible indicates a paucity of surface sites. Plasma albumin competes with thrombin for surface sites. Attempts to detect, by thrombin adsorption, the presence of free sites at 4.5 X 10-9M albumin or the displacement of bound albumin indicate an albumin-site association contrast greater than 1.6 X 10(9). Cross-linked poly(methyl acrylate) bead surface has U less than 5 X 10(10). In contrast to acrylic resins are silicone gum, polypropylene, and polyisobutylene, for which U ranges from 15 to 20 X 10(11). Analysis as above indicates that sites are of frequent occurrence. Material composition suggests that thrombin can interact with nonpolar groups. Further characteristics of low-energy surfaces are that progressive surface denaturation is small and there is a large variance between nominally equivalent configurations.


Mathematical models for the release of drugs from matrix tablets.

A previously derived cubic equation describing the release of drugs from matrix tablets is compared with a more complex equation recently presented. Using data obtained from cylindrical hydrocortisone matrix tablets, it is shown that the simpler cubic equation affords an equally acceptable description of drug release. Because the cubic equation contains a release-rate constant Kr that is independent of tablet shape, a congruency between this equation and other historical models of diffusion may be demonstrated. It is suggested, given the complexity of drug disposition in vivo, that simplicity in modeling is a desirable goal.


Defects in failed stems of hip prostheses.

A study has been made of the fractures and associated material from 34 prostheses broken in service. All the fractures appear to be of the fatigue type. Search by metallographic analysis for cracklike generating defects in material near the fracture reveals such conditions as abnormally coarse crystals, large nonmetallic inclusions, large inclusion population, undissolved master alloy particles, abnormal carbide segregation, interdendritic segregation, shrinkage, and gas porosity. Recommendations are made for the choice of metallic alloys for femoral stem application.


A novel technique for studying the adsorption of plasma proteins on hydrophobic surfaces.

A method is shown for deducing the surface area of a pendant drop from the same profile photograph as is normally used to determine interfacial tension. Manipulation of such drops by a micrometer syringe then enables the pendant drop to be used as a surface balance for studying adsorption from bulk solutions. Results are given for the compression of films from solutions of albumin, gamma-globulin, fibrinogen, albumin-gamma-globulin mixture and albumin-gamma-globulin-fibrinogen mixture, and from serum, plasma, and blood at the isooctane-buffer interface. It is suggested that gamma-globulin and fibrinogen films are unfolded at the interface but that albumin films are different in that they consist of an inner, unfolded layer and an outer, globular layer. A film from serum resembles that from gamma-globulin alone, and not that from albumin-gamma-globulin mixture, whereas a film from plasma resembles that of fibrinogen at low compression and that of albumin-gamma-globulin-fibrinogen mixture at high compression. A film from blood is shown to resemble that from plasma.


A high-modulus polymer for porous orthopedic implants: biomechanical compatibility of porous implants.

A high-modulus polymer, polysulfone, was evaluated as a porous bone implant material. The bone ingrowth into canine cortical pellets of sintered polysulfone particles was assessed by microradiography and histology. The shear strength of the porous polysulfone-bone interface was determined by push-out and pull-out tests of cortical and trochanteric implants, respectively. Results indicated that the bone ingrowth into porous polysulfone specimens proceeded in such a fashion as to mimic the normal repair at the site. Mechanical testing of cortical and cancellous implants revealed that the interfacial shear strength of the porous polysulfone-bone composite was similar to that achieved using porous metals.


Antibiotics in acrylic bone cement. In vitro studies.

The effect of combining gentamicin and cephalothin with Surgical Simplex bone cement was tested in vitro. Experiments were designed to determine (1) quantitative elution rates and period of time the antibiotics were eluted and (2) effective bactericidal qualities. Both antibiotics were eluted from polymerized acrylic cement in large quantities within 24--48 hr after coming in contact with fluid and continued to be released in smaller amounts for 21 days to more than 175 days. This varied with the antibiotic, concentration of antibiotic in the cement, surface area of cement, and volume of elution fluid. Quantitative determinations of numbers of bacteria in broth cultures demonstrated that sufficient quantities of antibiotics were released to be bactericidal to microorganisms within 45 min to 20 hr. This was dependent on the type of antibiotic, type of bacteria, concentration of antibiotic in the cement, and time of contact.


Adsorption of plasma proteins on hydrophobic surfaces. III. Serum, plasma, and blood.

Liquid-air and liquid-liquid interfaces were used as models for the liquid-solid system of plasma proteins and hydrophobic surfaces in the study of adsorption of serum, plasma, and blood onto these surfaces. The interfacial tension is determined for three phases: air, methylene iodide, and isooctane. Curves of interfacial tension versus time for the various systems are given from which a triple-intersection point, where the protein solution is in equilibrium with each surface, is found. It is shown that albumin, gamma-globulin, and a mixed solution of these at in vivo concentrations behave in characteristic and constant manners at the three interfaces of air, methylene iodide, and isooctane. A range of synthetic surfaces which have constant behavior at equilibrium is deduced and it is concluded that any soft tissue response differences between such surfaces could not be the result of albumin or gamma-globulin.


Corrosion behavior of cast and forged cobalt-based alloys for double-alloy joint endoprostheses.

An ideal combination of mechanical and corrosion properties of long-term implants such as joint endoprostheses has yet to be found. Besides being resistant to pitting and crevice attack, which can lead to corrosion fatigue and stress corrosion cracking failures, the implant material must be highly resistant to wear and abrasion. Two cobalt-based alloys, wrought CoNiMoTi and air-cast CoCrMo, were subjected to a number of selected in vitro electrochemically and chemically accelerated corrosion tests in chloride-containing solutions with wrought AISI-316L used as a reference alloy. A limited number of immersion tests in FeCl3 and acidified FeCl3 solutions were also conducted. It is found that the mechanical properties of wrought CoNiCrMoTi alloy qualify it as a substitute for cast CoCrMo alloy and wrought AISI-316L in anchorage shaft production for all types of joint endoprostheses. Wrought CoNiCrMoTi has a higher resistance to fatigue cracking compared with cast CoCrMo and is as resistant to selective corrosion phenomena such as stress corrosion cracking.


Partial or complete resection of the hemipelvis. An alternative to hindquarter amputation for periacetabular chondrosarcoma of the pelvis.

After excision of the hemipelvis through a combined subnatal and anterior incision preserving the lower extremity, five patients with periacetabular chondrosarcoma, after a follow-up of three to six years, were able to walk with no support or with minimum lateral support and had resumed their preoperative occupations. None had either the instability that might have been anticipated or incapacitating discomfort from the neoarthrosis, and there was no evidence of spread or recurrence of tumor in any of the five patients. The procedure, which has the same basic indications as a hindquarter amputation, is a rewarding substitute for that mutilating operation, although the follow-up is too short and the series is too small to warrant conclusions as to the cure rate.


Resection and reconstruction for primary neoplasms involving the innominate bone.

Using described criteria for the selection of patients for excision or resection of tumors involving various portions of the innominate bone, as opposed to hemipelvectomy, fifty-seven out of the more than 200 patients evaluated were judged to be candidates for a curative procedure. Of these, twenty-five were selected for hemipelvectomy and thirty-two, for non-amputative procedures. Depending on the location and extent of the lesion as determined by complete preoperative work-ups, three types of procedures were performed singly or in combination:(1) wide excision or radical resection of the iliac wing; (2) periacetabular wide excision or radical resection; or (3) wide excision or radical resection of the pubis. Reconstruction was accomplished when the hip joint was excised by fusion or the creation of a pseudarthrosis either medially in relation to the pubis or laterally in relation to the ilium or wing of the sacrum. The results after follow-ups of one to seventeen years were assessed in terms of the immediate goals of surgery, control of the disease, and function. The findings were as follows: With the preoperative assessment and operative techniques described, an oncologically adequate procedure was performed in two-thirds of the cases. In the remaining cases, the adequacy of the procedure was compromised by poorly planned biopsies, occult microextensions, and surgical errors. The recurrence rate was high after the inadequate procedure (100 per cent) and low (4 per cent) after the adequately accomplished procedures. Function was nearly normal when the hip joint was preserved. If the hip joint was removed and fusion was obtained, the results were good, but fusion was obtained in only 50 percent of the cases in which it was attempted. If the hip joint was removed and pseudarthrosis resulted, the results ranged from good to poor. Sciatic-nerve involvement necessitating resection of the nerve was not a contraindication to a non-amputative procedure.


Reconstruction of the pelvic ring following tumor resection.

Two patients with chondrosarcoma of the periacetabular region were treated by local resection and reconstruction of the pelvic ring and hip, implanting a Charnley-Mueller total hip replacement and reconstituting the pelvic ring with cement reinforced by Küntscher rods and heavy Kirschner wires. The first patient, in whom adequate resection was accomplished, was well five years later and was able to walk with a mild lurch and without a cane except when walking long distances on rough ground. Despite a fall causing a fracture of the ipsilateral tibial plateau at two years and a fatigue fracture of the ipsilateral ischium which healed at 4.5 years, the implant had remained intact. In the other patient, for whom hemipelvectomy was advised but who refused it, the resection was not adequate and although he was able to walk with a cane for short distances postoperatively, he died of metastases at two years. Based on these two cases, it would appear that reconstruction of the pelvic ring and hip may be feasible in a few very carefully selected cases of periacetabular tumors.


The Jones fracture revisited.

Twenty-two patients with twenty-three fractures of the proximal part of the diaphysis of the fifth metatarsal, an injury originally described by Jones, are reported. The average follow-up was 3.5 years. Delayed union occurred in twelve (66.7 per cent) of eighteen patients treated conservatively. Four patients were operated on within two weeks of injury. The clinical picture in nine of the twenty-two patients suggested that the injury was a stress fracture. Force-platform analysis in eleven cases confirmed that the injury results from vertical and mediolateral forces concentrated over the fifth metatarsal and that it is not caused by inversion. Thirteen of the twenty-three fractures were in young athletes, often occurring during training and causing significant disability. In this group of patients, and in a select number of recreational athletes and non-athletes with delayed union, intramedullary screw fixation of the fracture is advised.


Arthroscopy of the knee. Evaluation of an out-patient preocedure under local anesthesia.

In 228 knees submitted to arthrotomy following arthroscopy, there were eleven diagnostic errors in those knees examined under general anesthesia and five diagnostic errors in those knees examined under local anesthesia with a diagnostic accuracy of 91.1 and 95.2 per cent, respectively. Therefore, we concluded that accuracy is not diminished by performing arthroscopic examinations under local anesthesia on ambulatory out-patients. In 297 knees examined under local anesthesia, no complications were encountered. In 282 knees examined under general or spinal anesthesia, there was one pulmonary embolus and two significant hemarthroses. Except when there are specific overriding considerations, local anesthesia is preferable in the performance of diagnostic arthroscopy.


Arthrodesis in failed total knee replacement.

Compression arthrodesis can be used as a salvage procedure in failed total knee replacement. In fourteen patients so treated, the fusion failed in five. As regards the cause of failure, the most important factor was technical inadequacy of the arthrodesis procedure; that is, the required stable fixation and solid bone-to-bone contact. In procedures that we considered technically adequate, there was fusion in seven of the nine knees. The fusion seemed to be delayed in these patients, averaging 5.3 months (range, two to twelve months). Fixation with a double clamp seemed to be more effective than with a single clamp.


Hoke triple arthrodesis.

One hundred and nine cases of the Hoke triple arthrodesis which were done between 1945 and 1974 are reviewed. The results of the procedure are similar to other series insofar as correction of the deformity and the non-union rate are concerned. There was, however, an over-all incidence of aseptic necrosis of the talus of 6.5 per cent. A change in technique, in which the origin of the artery to the tarsal canal was left intact, has eliminated this complication. Excessive stress on the ankle joint as a result of the triple arthrodesis, although theoretically a problem, was not significant during follow-up of these patients. In the fourteen patients with myelomeningocele, there was acceleration of degeneration of the ankle joint, but Charcot joints did not develop.


Results of reduction and stabilization of the severely fractured thoracic and lumbar spine.

From 1962 to 1976, ninety-five patients with fracture-dislocations of the spine were treated with Harrington instrumentation and fusion within ninety days of injury. This report presents the results of this procedure related to reduction, stabilization, return of neural function, and total hospital stay. Mean follow-up was twenty-one months. Reduction and stabilization were attained without a substantial number of complications, but no more return of neural function in the patients was evident than has been reported in the literature for patients treated with postural reduction and bed rest. Total hospital stay averaged 107 days from day of injury.


Congenital thoracic lordosis.

The rare spinal deformity of congenital thoracic lordosis is illustrated in five patients. Often this deformity is progressive and may cause severe pulmonary deficits and death. In the two patients most recently treated, early spine fusion (one anterior and one posterior) showed promising results--halting of the progression of deformity and even some amelioration of the lordosis. In two patients in whom active attempts were made to correct the lordosis, the complications were severe--one patient died and quadriparesis developed in the other.


Osteotomy for tibia vara in Paget's disease under cover of calcitonin.

Five women with Paget's disease of the tibia were seen with pain in the knee, ankle, or both, as well as with tibial bone pain. All had tibia vara and internal torsion of the tibial shaft. Osteotomy to correct the deformities was preceded by a course of calcitonin which relieved the bone pain but did not relieve the articular pain. Relief after satisfactory correction of the tibial deformity was achieved in all patients. Calcitonin effectively minimized bleeding at the osteotomy site and complications were not encountered.


Arterial hypertension induced by femoral lengthening. A canine model.

In a canine experimental model, femora were lengthened 2.5 to 3.0 centimeters over a one-minute period. Systolic blood-pressure elevation averaged forty millimeters of mercury and diastolic blood-pressure elevation, twenty-two millimeters of mercury. The average peak arterial pressure was 193/115 millimeters of mercury. The effects of sympatholytic agents on the experimental model were tested. Alpha-adrenergic blockade by phenoxybenzamine, total-body catecholamine depletion by reserpine, and sympathetic ganglionic blockade by trimethaphan camphorsulfonate all inhibited the rise in arterial pressure, but beta-adrenergic blockade by propranolol did not. A differential spinal anesthetic with Xylocaine solution dramatically decreased the elevation in arterial pressure.


Treatment of unicameral bone cysts by curettage and packing with plaster-of-Paris pellets.

Long-term follow-up of twenty-six patients with unicameral bone cysts treated by curettage and packing with plaster-of-Paris pellets showed a recurrence in only two cases and no serious complications.


Treatment of orthopaedic infections with electrically generated silver ions. A preliminary report.

Electrically generated silver ions have been shown previously to be a potent antibacterial agent with an exceptionally broad spectrum as indicated by in vitro testing. The present study reports on clinical experience using electrically generated silver ions as adjunctive treatment in the management of chronic osteomyelitis. Fourteen patients had fifteen treatment attempts: thirteen for chronic osteomyelitis of the tibia, one for acute and chronic pyarthrosis and osteomyelitis of the knee, and one for a chronically draining sinus after total hip replacement. Wound débridement, silver ion iontophoresis, and subsequent wound care (usually provided by the patient) resulted in control of the infection in twelve of the fifteen treatment attempts and in healing of the non-union after follow-up ranging from three to thirty-six months. The other three attempts led to two partial and one complete failure.


Genu recurvatum in spastic cerebral palsy. Report on findings by gait analysis.

Using high-speed motion pictures, electromyography, a dynamic piezoelectric force plate, and computer analysis of the data, the gait patterns of fifteen children, four to sixteen years old, with spastic cerebral palsy and genu recurvatum were analyzed to determine the mechanisms producing genu recurvatum and the effect of fixed-ankle below-the-knee orthoses. In all children the recurvatum during stance phase began when the tibia stopped moving forward and disappeared when tibial movement resumed. In six patients (Group I), excessive activity of the calf muscles in response to the increasing dorsiflexion moment about the ankle produced by the foot-floor reaction force arrested the forward motion of the tibia. In six others (Group II), the contraction of the calf muscles was not sufficiently strong to resist the dorsiflexion moment and the tibia moved forward until maximum dorsiflexion had occurred and then stopped. In both instances recurvatum was produced when the femur continued to move forward over the stationary tibia and an extension moment was produced at the knee. In no patient did activity of the knee flexors prevent recurvatum, which was eliminated only by resumption of forward movement of the tibia. This movement of the tibia was produced either by heel-off (Group I) or by sudden unweighting of the limb due to opposite heel-strike (Group II). In Group I, when tibial motion stopped in the first half of stance phase the position of the hip rapidly changed from flexion to extension and there was forward leaning of the trunk, while in Group II the change from hip flexion to extension occurred with backward leaning of the trunk. The fixed-ankle below-the-knee orthosis, by preventing excessive dorsiflexion and plantar flexion, produced more normal moments about all joints, especially the knee. In the three children (Group III) whose recurvatum was permanently corrected by the brace, no explanation for the improvement was evident in these studies.


Postural sway in amputees and normal subjects.

The postural sway of amputees was investigated quantitatively in fifty patients with above-the-knee amputations and in twenty-nine patients with below-the-knee amputations. The factors evaluated were: (1) the mean speed of sway: (2) the range of movement in the sagittal and coronal planes; and (3) the dependence on vision as indicated by the ratio of eyes-open to eyes-closed values. The results demonstrated that in amputees there is an increased dependence on vision for maintenance of the erect posture. Postural sway in patients with below-the-knee amputations was found to be significantly greater than that in those with above-the-knee amputations. Postural sway increased with age; but the visual dependence for the control of postural sway was independent of age.


Anterior cruciate ligament repair for rotatory instability: the Lindemann dynamic muscle-transfer procedure.

The Lindemann operation, originally performed in 1950 as a reconstructive procedure for the anterior cruciate ligament, was performed in the knees of eight patients suffering from rotatory instability. Although the instability persisted, as shown by roentgenographic and clinical examination, treatment with the tendon transfer appears to have provided dynamic stabilization. Seven of the eight patients so treated were subjectively improved, and they returned to sports or activities which they had not been able to perform prior to surgery.


Fractures of the odontoid process in young children.

We reviewed eleven patients less than seven years old with fractures of the odontoid process in an effort to establish a more standard form of treatment for the injury and to determine what complications, if any, occur as a result of fractures of the odontoid process in pediatric patients. Our study showed that children with odontoid fractures that are recognized and treated promptly usually do well. The fracture can usually be reduced by passive manipulation or by the "hanging head technique". Support in the reduced position for two to three months in a Minerva jacket or halo cast should be long enough to permit healing. Our study suggests that fractures of the odontoid process in young patients almost always heal.


The spine in osteogenesis imperfecta.

A high incidence of spinal curvature, approaching 80 per cent in patients more than twelve years old, was found in a large series of patients with osteogenesis imperfecta. Curves showed early progression and a predictable pattern. In the age range between one and five years the incidence of scoliosis was 26 per cent; thereafter, it rose precipitously. The degree of curvature was greater in the older children. The Milwaukee brace was ineffective in the treatment of the scoliosis and the complications of its use were detrimental. In several patients it deformed the rib cage. It had little or no effect on the progression of the curve. The incidence and degree of curvature was related to the type of bone present. Patients with chest deformities and those who were not ambulatory seemed to have a predilection for scoliosis.


Survival and ambulation following hip fracture.

A retrospective analysis of the outcomes in 360 patients with fractures of the hip showed return to preinjury ambulatory status in 51 per cent. One year after injury 27 per cent of the patients had died and 22 per cent were non-ambulatory. For the first eight months after injury the death rate in the fracture population exceeded that of the population at large, while afterward it was the same. Factors that increased the probability of death and non-ambulation included advanced age, preoperative presence of cerebral dysfunction, and male sex.


Segmental fractures of the femur treated by closed intramedullary nailing.

Twenty acute segmental fractures of the femoral shaft were treated with closed intramedullary nailing. The severity of the soft-tissue damage and associated injuries necessitated careful preoperative care. The surgical technique and means of reduction are outlined. Follow-up of at least one year on all patients showed a 100 per cent rate of union with no infections. Major complications included splitting of the greater trochanter during nailing (one case) and an 11-degree valgus deformity (one case). Maximum shortening was 1.5 centimeters. Knee flexion averaged 135 degrees in these acute fractures. The results in this series indicate that closed intramedullary nailing is an excellent method of treatment for this difficult fracture.


Total hip arthroplasty in chronically dislocated hips. Follow-up study on the protrusio socket technique.

The so-called protrusio socket technique of total hip arthroplasty was shown to be very effective in a series of twenty-two chronically dislocated or subluxated hips. In seventeen patients with follow-up of sixteen to fifty-nine months, excellent results were obtained in all but two hips. Obesity and rheumatoid arthritis or other systemic causes of osteopenia are considered contraindications to the procedure.


Kinematics of the wrist. II. Clinical applications.

A reproducible method of measuring carpal height and carpal-ulnar distance and expressing them as ratios of these distances to the length of the third metacarpal, as reported in a previous paper9, was applied to serial roentgenograms of seventeen patients with rheumatoid disease and six patients with Kienböck's disease. These ratios provide an accurate expression of the extent of carpal collapse and of ulnar translation. However, once carpal collapse has occurred, the trajectory of hand motion becomes flattened, and, as a result, the carpal height ratio will vary as the wrist moves from maximum radial to maximum ulnar deviation. These ratios may be of use in predicting a patient's clinical course, but a larger clinical study is necessary before use of the measurements for this purpose can be recommended with assurance.


Acute ulnar neuropathy with fractures at the wrist.

Injury to the ulnar nerve associated with or following a fracture at the wrist is unusual. Three cases of this association are presented. Previously reported cases have been reviewed. The associated lesions tend to occur in young adults after higher-energy trauma and more dorsal displacement of the distal radial fragment than are generally seen in patients with Colles fractures. The return of function in the three wrists was related to the promptness and accuracy of reduction of the fracture and to the decompression of the ulnar nerve in Guyon's canal. We present a regimen for treatment of this injury stressing accurate reduction of the fracture and early surgical decompression when necessary.


Numerical assessment of bone scintigraphy in primary bone tumors and tumor-like conditions.

Fifty-four tumors or tumor-like conditions in bone were studied by numerically assessed 99m-technetium polyphosphate scintigraphy. The uptake was expressed as a ratio of the uptake in the tumor region to the uptake in a corresponding region in the contralateral part of the body. In the malignant tumors there was marked variation in the uptake within each individual tumor group, a variation that rendered a differential diagnosis impossible. In general, the uptake was fairly high in malignant tumors and lower in benign ones. A ratio below 1.5 suggested the likelihood that the lesion was benign. The ratios also varied considerably with the site of the tumor and the patients' ages. Relatively low ratios were found for tumors of the trunk and for juxta-articular tumors in children, whereas higher and more varied ratios were observed in tumors of the peripheral skeleton in adults. It is concluded that numerically assessed scintigraphy is not a useful supplement to other methods used for diagnosing bone tumors.


Results of surgical treatment for non-union after high tibial osteotomy in osteoarthritis of the knee.

In a series of 280 high tibial osteotomies performed for osteoarthritis of the knee between 1969 and 1975, there were ten cases of pseudarthrosis, an incidence of 3.6 per cent. These ten knees (and an additional two that were referred to us) were reoperated on. In most cases the pseudarthrosis was resected and stabilized with the Charnley transfixation-compression method. Other procedures involved resection without compression (one knee), compression blade-plate fixation, and arthroplasty with a hinge endoprosthesis. All osteotomies healed eventually with the knee in satisfactory position. In spite of the initial non-union and repeated operation, all twelve patients eventually had satisfactory correction of the preosteotomy deformity, and none had a loss in walking ability. All but two patients had freedom from pain. We concluded that patients with non-union following high tibial osteotomy for osteoarthritis of the knee should undergo resection of the pseudarthrosis and transfixation compression as the treatment of choice. Endoprosthetic replacement then can be used as a salvage procedure if it is needed.


Neurofilament proteins of rat peripheral nerve and spinal cord.

Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.