Transport of amino acids in renal brush border membrane vesicles. Uptake of L-proline. The findings (a) stereospecific counter transport; (b) equilibrium uptake of L-proline decreased with increasing medium osmolarity; and (c) L-proline and Na+ were taken up into identical intravesicular spaces, indicate that uptake of L-proline by rabbit renal brush border membranes represents transport into membrane vesicles, not surface binding to the membrane. An Na+ gradient between extravesicular and intravesicular media stimulated the initial rate of L-proline uptake about 10 times. Accumulation of the imino acid was maximal at 2 min, then decreased until the equilibrium level was attained. At the peak of this "overshoot" uptake of L-proline was 3-fold greater than the final equilibrium value. These results suggest that the electrochemical Na+ gradient drives the transient movement of L-proline into the membrane vesicles against its concentration gradient. Stimulation of L-proline uptake was specific for Na+. Increasing the Na+ gradient lowered the apparent Km for L-proline. Efflux of L-proline from the membrane vesicles, like uptake, showed stimulatory effects when the Na+ gradient and L-proline were on the same side, and inhibitory effects when the Na+ gradient and the imino acid were on opposite sides of the membrane. Uptake of L-proline, at a given concentration, reflected the sum of contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at 4 mM L-proline. The independent system did not saturate but may represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations the rate of the Na+ gradient-dependent uptake was 5 times that in the absence of the gradient. In K+-loaded vesicles, valinomycin, but not nigericin, enhanced the Na+ gradient-dependent uptake of L-proline. Gramicidin diminished uptake. These findings indicate that the Na+ gradient-dependent transport of L-proline is an electrogenic process and suggest that the membrane potential is a determinant of L-proline transport. The Na+ gradient-dependent rate of L-proline uptake was strongly inhibited by other imino acids, suggesting that L-imino acids have a common transport system. Glycine and neutral amino acids inhibited the rate of L-proline uptake moderately, acidic amino acid and D-glucose were very weak inhibitors, and basic amino acids were without effect. In the absence of Na+, the rate of L-proline uptake was independent of the presence of other amino acids. These findings indicate that the brush border membrane is a site of amino acid recognition during vectorial transepithelial transport. It is proposed that imino and neutral amino acids inhibit transport of L-proline by competitive interaction with the L-proline "carriers" in addition to competition for the electrochemical Na+ gradient or membrane potential. The findings are relevant to the understanding of genetic amino acid transport disorders, such as iminoglycinuria.


The covalent structure of cartilage collagen. Evidence for sequence heterogeneity of bovine alpha1(II) chains. During studies on the amino acid sequence of bovine nasal cartilage collagen, the cyanogen bromide peptide alpha1(II)-CB11 was degraded to smaller peptides with trypsin. One of the tryptic peptides, T5, which contained 39 residues was shown by amino acid and sequence analyses to occur in a predominant form that contained glutamine at position 5 and in a second form with leucine at this site. In addition to the heterogeneity at this position, amino acid analyses of five different preparations revealed that the peptide with leucine contained a seryl residue not found in the major form. Sequence heterogeneity at a third position of alpha1(II) was demonstrated by the isolation of a hexapeptide (T2) from the trypsin digest of alpha1(II)-CB11 which contained 0.21 residue of alanine and 0.77 of leucine. Both the leucine and alanine of T2 were removed after the second cycle of subtractive Edman degradation. These data show that at least two types of alpha1(II) chains, designated as alpha1(II)Major and alpha1(II)Minor, exist in bovine nasal cartilage. Further considerations suggest that these two chains are probably not variants derived from allelic genes but are the products of separate genes.


Mechanism of induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human leukocytes. Incubation of leukocytes in buffer alone devoid of lipoproteins does not lead to the induction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, but incubation of these cells in lipid-depleted serum, abetaliprproteinemic serum, or lipoprotein-deficient serum (d greater than 1.21) leads to sterol loss from the cells and the activation of sterol synthesis from acetate. The latter was shown previously to be proportional to the HMG-CoA reductase levels in the cells (Fogelman, A. M., Edmond, J., Seager, J., and Popják, G. (1975)J. Biol. Chem. 250, 2045-2055). Sterol loss occurs from normal and heterozygous familial hypercholesterolemic leukocytes within 15 min in lipid-depleted serum. Since induction of HMG-CoA reductase activity is not detectable until after the leukocytes have been incubated in the lipid-depleted serum for at least 3 h (Fogelman et al., see above), sterol loss clearly precedes the induction of the enzyme. In six out of six experiments, the nonisotopic sterol content of leukocytes incubated in lipid-depleted serum was equal to or lower than that of the same leukocytes incubated in full serum. This occurred at a time when the leukocytes in the lipid-depleted serum were incorporating 4 to 5 times more [14C]acetate into sterols than the same leukocytes in full serum. This strongly suggests that the induction of the reductase was a compensatory mechanism for sterol loss. Incubation of leukocytes in buffer, or buffer plus lecithin dispersions, or buffer plus albumin did not lead to sterol loss or induction of the reductase, but incubation in buffer and albumin together with lecithin dispersions caused sterol loss into the medium and the activation of sterol synthesis from acetate. It is concluded that a phospholipid-protein-cell interaction, which produces sterol loss, is necessary to induce the reductase in leukocytes. A close correlation between sterol loss and total sterol synthesis (a function of HMG-CoA reductase activity) was demonstrated in normal and heterozygous leukocytes incubated in a variety of incubation media (r = 0.95; p less than 0.005). Heterozygous leukocytes taken fresh from the blood contained no more cholesterol than the leukocytes of their age and sex-matched controls, despite the marked difference in their serum cholesterol concentrations. It is proposed that the abnormality in familial hypercholesterolemia can be accounted for by an abnormal efflux of cholesterol from heterozygous cells.


Study of RNase A mechanism and folding by means of synthetic 63-residue analogs. A 63-residue RNase A analog containing residues 26 to 35 then alanine, 41 to 59 and 73 to 84 then glycine, 100 to 110 then glycine, and 117 to 124 was synthesized by the solid phase method. The deletions comprised ordered (an alpha helix, parts of the beta sheet) and less ordered structures including 27 of the 56 residues invariant in mammalian ribonucleases. The synthetic 63-residue analog was cleaved from the insoluble support with liquid HF, reduced-reoxidized, fractionated by gel filtration, and purified further on an affinity column specific for the active site fold of RNase A. It had an activity of 8 to 14 per cent in the transphosphorylation step using poly(C) and poly(U) as substrates. It also had low synthetic and hydrolytic activity (0.2 per cent) and showed RNase A-like specificity toward the substrates tested. This indicated that all residues essential for substrate binding and catalysis were present and that their relative positions in the three-dimensional structure were probably very similar to those in native RNase A. Therefore, structure-function studies with the 63-residue RNase A analog should allow conclusions about the mode of action of the natural enzyme. As a first step in this direction, lysine 41 which is believed to be important for catalysis was replaced in the 63-residue analog by tyrosine or glutamine. The resulting (Tyr-41)- and (Gln 41)-63-residue analogs were also bound by the affinity column and had the same substrate specificity as native RNase A. They differed from each other, from the (Lys 41)-63-residue analog, and the 124-residue natural enzyme only with respect to the relative rates of the catalyzed reactions. Thus, lysine 41 does not seem to be essential for the functioning of RNase A.


Topography of the external surface of the human red blood cell membrane studied with a nonpenetrating label, [125I]diazodiiodosulfanilic acid. The topography of the external surface of the human red cell membrane has been studied using an impermeant radioactive probe, [125I]diazodiiodosulfanilic acid, which binds covalently to protein groups of the membrane following reaction with intact cells. The pattern of labeling was assessed by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis followed by sequential analysis of single gels for carbohydrates (by staining with the periodic acid-Schiff (PAS) reagent), for proteins (by staining with Coomassie blue), and for radioactivity (by counting gels sliced in 2-mm segments). The radioactive probe bound to membrane polypeptides with apparent molecular weights of 94,200, 58,100, and 46,500 (Peaks A, B, and C, respectively). Peak A co-migrated with a small periodic acid-Schiff-positive band and protein Band 3 (nomenclature of Steck) (Steck, T.L. (1974)J. Cell Biol. 62: 1-19). Peak B migrated with protein Band(s) 4.5 slightly ahead of the major membrane glycoprotein (PAS-1). Peak C migrated like glycoprotein PAS-2 and protein Band 5, the actin-like, water-soluble membrane protein. In contrast to lactoperoxidase iodination and a number of other probes, [125I]diazodiiodosulfanilic acid reacted minimally with the major membrane glycoprotein, glycophorin. When it was reacted with isolated ghosts, all molecular weight classes of polypeptides were labeled. Treatment of labeled cells with neuraminidase or trypsin altered the glycoprotein staining pattern, but not the radioactive peaks. On the other hand, Pronase eliminated the Mr=94,200 radioactive peak, diminished the other two radioactive peaks, and profoundly changed the glycoprotein and protein staining patterns. Treatment of the membranes of labeled cells in a low ionic strength alkaline medium did not alter radioactive peaks and demonstrated that Peak C differed from the actin-like membrane protein. A nonionic detergent, Triton X-100, solubilized all radioactive components. The studies have defined the binding of [125I]diazodiiodosulfanilic acid to external proteins of the human red cell membrane. Its pattern of reaction differs quantitatively and qualitatively from other commonly used reagents, and it provides a useful additional vectorial probe for the study of membrane topography. Its reactions provide further evidence of the organizational complexity of the red cell membrane and emphasize the fact that interpretation of information derived from the use of membrane probes must take into account the differences resulting from the properties of the probing reagents themselves.


Effect of ribosomal proteins on synthesis and assembly of preribosomal particles in isolated rat liver nuclei. Ribosomal proteins are rapidly taken up by isolated rat liver nuclei. The proteins are localized mainly in the nucleolus and are found associated with a nucleolar 80 S particle containing newly synthesized 45 S RNA. Ribosomal proteins in the nucleoplasm were associated with particles containing 18 and 28 S RNAs. In the absence of ribosomal proteins in the incubations, there was a decrease in the amount of newly synthesized 45, 18, and 28 S RNAs and an increase in low molecular weight RNA in both the nucleolus and nucleoplasm. This in vitro system appears to be useful for studies on the formation of ribosomal particles in the nucleus.


Identification of glycoproteins and proteins at synapses in the central nervous system. Synaptic plasma membranes, synaptic junctions, and postsynaptic densities have been isolated from rat brain, the proteins resolved by polyacrylamide gel electrophoresis, and the glycoproteins identified. The synaptic junction is composed of a spectrum of polypeptides which range in Mr from 13,000 to 250,000. The overall pattern is similar to synaptic plasma memranes; however, the relative proportions of the polypeptides are distinctive. The postsynaptic density fraction consists primarily of one band with an Mr of 52,000. Polypeptides with an Mr of 55,000, and another five of higher Mr, make up the remaining protein. The polypeptides of the postsynaptic density fraction must be reduced with mercaptoethanol in order to permeate the polyacrylamide gel. Therefore, postsynaptic density proteins are cross-linked by disulfide bonds into supramacromolecular aggregates. Glycoproteins which bind concanavalin A were identified in synaptic junctions by studying the binding of 123I-concanavalin A directly to the polypeptides resolved on the polyacrylamide gels. Only four bands, each with an Mr greater than 95,000, bind concanavalin A. In contrast, the pattern of concanavalin A-binding polypeptides in synaptic plasma membranes is distinctive and more complex. In the postsynaptic density fraction, most of the concanavalin A binding occurs to a glyco-component which migrates at the dye front. These data, together with previous cytochemical data using concanavalin A-ferritin conjugates, indicate a limited and select group of high Mr concanavalin A glycoproteins resides within the synaptic cleft of asymmetric type synapses. Whereas a select group of polypeptides bind concanavalin A, all polypeptides resolved in the synaptic junction fraction are glycoproteins and contain galactosyl or galactosyl-like residues, since they label with tritiated borohydride following galactose oxidase treatment. This suggests that the carbohydrate composition of individual glycoproteins is different.


Effect of the A and B protomers of choleragen on release of trapped glucose from liposomes containing or lacking ganglioside GM1. Liposomes containing trapped glucose were used to examine the interaction of the A and B protomers of choleragen with ganglioside GM1 and lipid model membranes. The B protomer (choleragenoid) was as effective as choleragen in causing release of trapped glucose from liposomes containing GM1; the A protomer did not release glucose from GM1 liposomes. Neither choleragen nor the A or B protomers caused release of trapped glucose from glycolipid-free liposomes. Anti-choleragen and complement, however, caused release of trapped glucose from ganlioside-free liposomes previously incubated with the A protomer but not from those incubated with the B protomer or choleragen. These results suggest that the A protomer, but not intact choleragen or B protomer, bound to ganglioside-free liposomes. Presumably, the A protomer must be freed of the constraints present in the intact choleragen in order to interact with the liposomal model membrane system.


Resolution of beta-adrenergic receptor binding and adenylate cyclase activity by gel exclusion chromatography. The frog erythrocyte membrane provides an excellent model system for the study of beta-adrenergic receptor-adenylate cyclase interactions since it possesses an adenylate cyclase enzyme which is very responsive to catecholamines. The purpose of these studies was to evaluate directly whether the functions of receptor binding and adenylate cyclase activity are carried out by a single macromolecule or separable molecular entities. Obtaining this information is a first step in understanding at a molecular level how receptor binding is "coupled" to enzyme activation. Binding and cyclase activities were solubilized from the frog erythrocyte membrane with digitonin and were observed to partition independently during gel exclusion chromatography in the presence of solubilizing detergent. This finding documents that the beta-adrenergic receptor and adenylate cyclase enzyme are, in fact, separable macromolecules. Under the particular experimental conditions employed, the elution of beta-adrenergic receptor binding on Sepharose 6B was not altered by the absence or presence of beta-adrenergic agonist or antagonist ligands or by exposure of the membranes prior to solubulization to the guanyl nucleotide analog, guanyl-5'-yl imidodiphosphate.


Bacterial luciferase subunits are synthesized in equal quantities. Synthesis of luciferase, an alpha beta dimer, occurs during a relatively short period of time near the end of exponential growth of Beneckea harveyi. The rates of synthesis of the individual alpha and beta chains of luciferase are compared by quantitating the molar ratio of total cellular alpha to that of beta at different growth stages. The addition of exogenous alpha or beta subunit to crude lysates of cells taken prior to and during the luciferase induction produces no increase in luciferase activity. This result, together with previous evidence that there are no antigenically cross-reacting precursors, allows us to conclude that luciferase alpha and beta chains are synthesized pari passu, i.e. in equal proportions, and exist primarily in the alpha beta dimeric form. It is also shown that insoluble sedimentable cellular materials contain no detectable luciferase subunits.


Radial club hand. A continuing study of sixty-eight patients with one hundred and seventeen club hands. Based on a review of the embryology, genetics, and anatomy of radial club hand, it is suggested that damage to the apical ectoderm on the anterior aspect of a developing limb bud leads to the deformity. Study of the families of thirty-five children with radial club hand suggested that the condition is not genetically patterned. The anatomical findings and associated congenital abnormalities in the cases known to be related to thalidomide and in those in which thalidomide was not a factor were similar except that the incidence of other skeletal deficiencies was higher in the thalidomide group. Thirty-one of the 117 radial club-hand deformities (in sixty-eight patients) under my personal supervision were treated by centralization of the carpus on the ulna with satisfactory improvement of the deformity. In three cases wrist deformity recurred mainly in a volar direction, apparently the result of muscle imbalance. No significant impairment of ulnar growth occurred and straightening of the wrist did not affect function adversely. Pollicization of the index finger was done on twenty-eight occasions. Although problems developed in the early cases, these can be avoided using the methods described and the operation can improve both function and appearance. A scheme of management is recommended.


Surgical repositioning of the medial collateral ligament. An anatomical and mechanical analysis. Analytical techniques using multiple-exposure roentgenograms were employed to investigate surgical repositioning of either the femoral or the tibial attachment of the medial collateral ligament. The motion of the femoral attachment of the ligament with respect to the tibial attachment was used to compute the changes in length of the borders of the ligament for normal knees and for knees with repositioned attachments. The results support the conclusion that when advancement of the medial collateral ligament is utilized in the treatment of medial instability, optimization is accomplished by distal and anterior advancement with the knee in 30 degrees of flexion. Femoral displacement (proximal realignment) or tibial displacement at knee-flexion angles greater than 45 degrees is not recommended.


The diagnosis of chondromalacia by double contrast arthrography of the knee. The patellar and articular cartilages of the femoral and tibial condyles can be demonstrated well by double contrast arthrography, and abnormalities of the cartilage can be seen. A comparison of surgical findings with the preoperative arthrogram was made for 100 patients, and the diagnosis of chondromalacia of the patella and condyles had an accuracy of about 90 per cent.


Post-traumatic instability of the metacarpophalangeal joint of the thumb. Of eithty-six patients with collateral ligament instability of the metacarpophalangeal joint of the thumb, sixty-six (77 per cent) had ulnar and twenty (23 per cent) had radial instability, while in addition twenty-four had fractures and twenty-nine had volar subluxation of the proximal phalanx. Of the sixty-nine patients operated on, sixty-five had an abnormality of the ulnar or radial collateral ligament proper or of its attachment; two, subluxation of the extensor pollicis longus; and two, stretching of the adductor expansion. Stability was restored by collateral ligament repair or reconstruction, by fixation of a fracture fragment with a Kirschner wire, or by arthrodesis in all but five thumbs. Of the five patients who did not have stability of the metacarpophalangeal joint following surgery, three had had the collateral ligament sutured more than three weeks after injury and two had had reconstruction of the collateral ligament using a tendon graft. Early surgical treatment is recommended for all patients with post-traumatic instability of the metacarpophalangeal joint greater than 45 degrees and for those with volar subluxation of the proximal phalanx or a displaced fracture of the base of the proximal phalanx. Collateral ligament repair is indicated for patients operated on within three weeks of injury, and reconstruction of the ligament by means of a tendon graft is recommended for those treated more than three weeks after injury.


Fusion of the first metacarpotrapezial joint for degenerative arthritis. During a twelve-year period, twenty-eight patients (thirty thumbs) were treated for painful idiopathic arthritis of the metacarpotrapezial joint of the thumb by fusion. Failure of fusion occurred in two thumbs, and in both instances a solid fusion followed a second procedure. Fusion of the metacarpotrapezial joint did not predispose to painful arthritis of the trapezioscaphoid joint, even in patients with pre-existing roentgenographic evidence of minor degenerative changes in this joint. The results after long-term follow-up were gratifying, the patients having painless and stable thumbs with excellent strength. Although patients noted a minor loss of thumb motion, they did not consider this a problem. Fusion is a satisfactory procedure for patients who need or desire a strong, painless thumb, and seems especially worth while in the dominant thumb when both thumbs require surgical treatment.


Biomechanical analysis of static forces in the thumb during hand function. Using a three-dimensional analysis, the internal forces in the joints and soft tissues of the thumb during pinch and grasp were calculated. To do this, mechanical equivalents were ascribed to the anatomical system, the joint orientation and tendon locations were determined from biplanar roentgenograms of five marked normal cadaver specimens, and the magnitudes of forces in the tendons, intrinsic muscles, joint contact surfaces, and constraining ligaments were calculated based on assumed loads applied to the tip of the thumb in various types of pinch and grasp. These results are the direct extension of a two-dimensional analysis that proved inadequate for the determination of static tendon and joint forces. Extrinsic and intrinsic tendons of the thumb were found to sustain forces of up to 10.0 and thirty kilograms during pinch, producing five kilograms of force at the thumb tip and forces of as much as fifty kilograms during grasp. The joint compression (contact) forces averaged three kilograms of force at the interphalangeal joint, 5.4 kilograms at the metacarpophalangeal joint, and 12.0 kilograms at the carpometacarpal joint during simple pinch (one kilogram of applied force). Compression forces of as much as 120 kilograms may occur at the carpometacarpal joint during strong grasp.


Atlanto-axial rotatory fixation. (Fixed rotatory subluxation of the atlanto-axial joint). In seventeen cases of irreducible atlanto-axial rotatory subluxation (here called fixation), the striking features were the delay in diagnosis and the persistent clinical and roentgenographic deformities. All patients had torticollis and restricted, often painful neck motion, and seven young patients with long-standing deformity had flattening on one side of the face. The diagnosis was suggested by the plain roentgenograms and tomograms and confirmed by persistence of the deformity as demonstrated by cineroentgenography. Treatment included skull traction, followed by atlanto-axial arthrodesis if necessary. Of the thirteen patients treated by atlanto-axial arthrodesis, eleven had good results, one had a fair result, and one had not been followed for long enough to determine the result. Of the remaining four patients, one treated conservatively had not been followed for long enough to evaluate the result, two declined surgery, and one died while in traction as the result of cord transection produced by further rotation of the atlas on the axis despite the traction.


Fracture-dislocations of the cervical spine. Instability and recurrent deformity following treatment by anterior interbody fusion. Anterior intervertebral-body fusion has been advocated by various authors for management of several varieties of fracture and fracture-dislocation of the cervical spine. This report is a retrospective study of sixteen patients, all of whom had a fracture-dislocation of the cervical spine, who were treated with an anterior dowel interbody fusion. All had postoperative instability with recurrence of angular deformity, and all sixteen were shown to have disruption of the posterior ligaments. Three patients had a progressive neurological deficit postoperatively. Thirteen patients required two or more operative attempts at stabilization. Anterior fusion should not be performed as primary surgical treatment for fractures of the cervical spine when there is either evidence of disruption of the posterior ligaments or a strong presumption that such disruption exists.


Reactive sclerosis of a pedicle and spondylolysis in the lumbar spine. Eleven patients were seen with reactive sclerosis and hypertrophy of one pedicle and lamina of a lumbar vertebra and a contralateral spondylolysis in the same vertebral segment. Differentiating the reactive sclerosis in this condition from that associated with osteoid-osteoma is necessary since excision of a sclerotic pedicle associated with contralateral spondylolysis will create painful instability. In asymptomatic patients observation is sufficient, while in those with symptoms not relieved by external immobilization, spine fusion, usually of three levels, is indicated.


Fixation of Dwyer screws for the treatment of scoliosis. A postmortem study. The strength of fixation of Dwyer screws in seven or eight vertebral bodies in each of six fresh osteoporotic cadavera was measured under the following conditions: with fixation with only the screw; with the staple-screw combination; with each of these modes of fixation combined with polymethylmethacrylate placed in the hole prior to insertion of the screw; and with the screw reinserted after failure and filling the hole with methacrylate. The strength of fixation of the screw-staple combination was increased almost twofold by the methacrylate, and after failure followed by reinsertion of the screw embedding it in methacrylate, strong fixation was restored.


Disappearing bone disease. A clinical and histological study. The case histories of three patients, a man and two boys, with disappearing bone disease are reported. The clinical, roentgenographic, and histopathological features are described in detail. Histologically, in the early stages of the disease, the vanishing bone is replaced by numerous wide engorged capillaries. Eventually the bone is replaced by dense fibrous tissue. All three patients were treated by radiotherapy. Histochemical studies performed in one case revealed strong acid phosphatase and leucine aminopeptidase activities in perivascular mononuclear cells (possibly pericytes), suggesting that these cells took part in the bone resorption.


Remodeling of the femoral neck after in situ pinning for slipped capital femoral epiphysis. During a period of thirty-six years, sixty-two patients with seventy slipped capital femoral epiphyses were treated by pinning in situ. Twelve of these patients, ten years and eight months to sixteen years and one month old, were treated for moderate to severe slipping by pinning in situ. After follow-ups ranging from two to seventeen years, all but two patients had satisfactory remodeling of the femoral head and neck and were asymptomatic. The two with incomplete or no remodeling had no symptoms. It was concluded that the effects of remodeling have been largely ignored and that pinning in situ when possible, followed if necessary by osteoplasty or osteotomy through the lesser trochanter, is a safe and effective treatment.


Isolated avulsion fracture of the tibial attachment of the posterior cruciate ligament. Twenty-one patients with isolated avulsion fracture of the tibial attachment of the posterior cruciate ligament were treated in our hospital during the period 1965 to 1976. Twelve of the patients had fresh injuries and nine, injuries with delayed union or non-union. Twelve of the twenty-one patients were treated conservatively and nine, by surgical repair by means of staple fixation. Detachment of the posterior horn of the meniscus with an avulsed and displaced bone fragment was found in five of the nine patients operated on. All the patients were followed for an average of four years and eight months. Satisfactory results were obtained in every instance.


Isolated subcutaneous nodules (pseudorheumatoid). Ten patients with isolated subcutaneous nodules histologically similar to rheumatoid nodules were studied. Because of the intense fibroblastic reaction in one such nodule, it was initially mistaken for a fibrosarcoma. The nodules tended to be multiple and recurrent, with eventual spontaneous regression, and they were not associated with any systemic illness. The etiology of these lesions is unclear but the possibility of subsequent development of systemic rheumatoid disease is remote.


Influence of positioning of prosthesis in total knee replacement. The early clinical results of geometric total knee arthroplasty were  compared with the position of the prosthetic device by a roentgenographic score system. It was noted that there is a statistically significant positive correlation between a good clinical result and a well positioned prosthesis. In addition, it was appreciated that perfect positioning of the device was difficult to obtain. We believe that the long-term clinical results, wear resistance, and resistance to prosthetic failure depend on correct positioning of the devices.


The bone-cement interface temperature during total joint replacement. Sets of thermoprobe measurements of the temperature of the bone-cement interface were made during twenty total joint-replacement procedures. Although the range of rise in temperature was wide, from 3 to 17 degrees centigrade, the highest temperature obtained was 48 degrees centigrade, which is well below the denaturation point (56 degrees centigrade) of proteins. Several factors, such as the presence of blood and moisture at the interface and the large surface area and poor heat conductivity of methylmethacrylate, prevent the interface from experiencing the high rise in temperature that occurs at the center of the polymerizing cement mass.


Neurological symptoms in achondroplastic dwarfs--surgical treatment. Fourteen patients who had neurological symptoms associated with achondroplastic dwarfism and who had spine surgery were analyzed, as were an additional twenty-nine cases reported in the literature. Four types of neurological patterns emerged, based on the onset, symptoms, and physical examination. Some etiological correlation between the anatomical lesion and the result was made in each category, and a reliable prognosis related to the pattern is now possible. Early and appropriately extensive surgery may well be recommended, based on the patients treated.


Acute Monteggia lesions in children.

Acute Monteggia lesions in children. Twenty-five consecutive acute Monteggia lesions in children were treated during a six-year period. The series included a very rare case of a Type-II Monteggia lesion not previously reported in children. All patients, except one, were treated by closed reduction. In two instances, after a successful reduction the unstable radial head was fixed with a percutaneous Kirschner wire inserted through the capitulum with the elbow flexed 90 degrees. This was done to avoid immobilizing the elbow in an acutely flexed position. On follow-up (average, thirty-four months), all the end results were excellent.


Habitual toe-walkers. A clinical and electromyographic gait analysis. Six children who were habitual toe-walkers were studied using electromyographic techniques. In the initial gait evaluation, the muscle synergy pattern was found to be abnormal during the toe-toe gait as well as during the heel-toe gait. After treatment with casts, each patient had a normal electromyographic pattern during heel-toe gait.


Use of the spiral coil membrane oxygenator during open heart surgery in infants and children. The spiral coil membrane oxygenator was used without an arterial line filter during short term total cardiopulmonary bypass in 50 infants and children. The compact oxygenator proved to be efficient and reliable, was easy to operate in a simple perfusion circuit, has a high flow rate to priming volume ratio and eliminated gaseous emboli. During perfusion, platelet counts and fibrinogen concentrations descreased 51 and 31% respectively. Coagulation studies in eight infants less than 15 kilograms perfused with the membrane oxygenator system did not differ from those in 7 infants perfused by a bubble oxygenator system.


Primary intracavitary cardiac tumors: a review of 11 surgical cases. During the past 14 years, 11 patients varying age from 27 to 68 years, had excision of primary intracavitary cardiac tumor: 7 had a left atrial myxoma, 1 a left ventricular myxoma, 1 a right ventricular myxoma, and 2 had a left atrial malignant mesenchymoma. Accurate diagnosis was made by angiocardiography in 8 patients. All patients underwent open heart excision of the tumor with the use of cardiopulmonary bypass. One of the 9 patients with a left atrial myxoma died postoperatively with recurrence of the tumor. The two patients with left atrial malignant mesenchymoma died 8 and 15 months after palliation. All surviving patients are asymptomatic without evidence of recurrence.


Post-infarction ventricular septal rupture: surgical considerations and results on five operated patients. Five patients were operated on for ventricular septal rupture following myocardial infarction. The mean time interval between occurrence of infarction and surgery was 37 days (from 4 to 54 days). At EKG, the infarction site was anteroseptal in two cases and postero-inferior in three. Progressive deterioration of cardiac function leading to cardiogenic shock was noticed in four patients. Four patients had cardiac catheterization before surgery: the mean pulmonary to systemic flow ratio was 2.5:1 (from 3.8:1 to 1.8:1); in each case L.V.E.D.P., pulmonary wedge and pulmonary arterial pressures were elevated. Left ventricular angiograms and selective coronariograms were obtained in three patients, demonstrating the presence of left ventricular aneurysm in all three. In two patients, repair was performed through right ventriculotomy; in patients with left ventricular aneurysm, a left ventriculotomy and aneurysm resection were performed. This technique seems to permit much clearer view of the rupture site. In all patients the V.S.D. was closed by a dacron patch, reinforced with teflon pledgets. Two of the five patients died in the early postoperative period. Both had been operated within the first week from the onset of the ventricular septal rupture, because of cardiogenic shock, refractory to medical therapy. The three survivors showed remarkable improvement after surgery and they are in functional class II at a mean follow up period of 20 months. Emphasis is put on factors influencing surgical treatments and prognosis of these patients.


Plasma heparin neutralizing activity in patients with valvular stenosis and prosthetic heart valves. Patients with mitral and aortic valvular stenosis had plasma heparin neutralizing activity (HNA) which were comparable to the control group. In contrast, plasma HNA was elevated in patients with prosthetic valves, particularly in the group with prosthetic aortic valves. Neither the type nor the size of the valve appeared to influence the HNA level. No correlation was evident between plasma HNA levels and a history of systemic arterial embolism. It remains to be determined whether elevated plasma HNA levels result from platelet aggregation on the valve surface, or from platelet lysis due to turbulence around the prosthetic valve.


Scanning-electron-microscopic and functional studies of oxygenator-membranes during long-term-perfusion. Various types and amount of deposits were noted on the surface of different membrane oxygenators during experimental long-term perfusion in sheep studied by SEM. They were present in spite of adequate heparinization. The deposition of blood material suggests, that the imperfection of the membranes by fabrication must be controlled and improved by oneself for use in long-term perfusion.


The role of primary and secondary profundaplasty in the treatment of vascular insufficiency. Profundaplasty has been performed on 58 limbs (45 primary and 13 secondary) in 45 men. Thirty-four of these had incapacitating claudication, 14 rest pain, and 10 either gangrene or ischemic ulceration. In 19 treated by profundaplasty alone there were no deaths but three subsequently had amputation for ischemic pain. In 39 with profundaplasty plus a proximal operative augmentation there were two (6.7%) operative deaths and one (3.4%) late death but only one extremity had to be amputated. Radionuclide flow studies confirmed physical and arteriographic findings. After profundaplasty alone and profundaplasty plus aortofemoral bypass there was moderate increase in calf blood flow but in only those with a patent superficial femoral did the flow studies return to normal. Profundaplasty is an important addition to the armamentarium of the vascular surgeon in dealing with arteriosclerotic insufficiency of the lower extremities.


The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii. The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter-conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell  cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide.


Autologous arterial grafts grown on polyester molds: observations of graft growth and patency in experimental animals. Tissue molds consisting of polyester mesh jackets covering silicone mandrils were implanted subcutaneously in dogs. When a sufficiently strong layer of connective tissue had formed around the molds they were excised and the mandrils removed. The process of tissue formation was observed and recorded. The forms were used as grafts in either the abdominal aorta or the femoral artery of the dog. Six grafts 10 mm inner diameter, six grafts 6 mm inner diameter and 26 grafts 3 mm inner diameter were implanted. After 4.5 months 80% of the 10 mm grafts, 83% of the 6 mm grafts, but only 4% of the 3 mm grafts remained patent. Histological studies at various intervals after grafting revealed gradual thickening of the neointima. The reason for graft failure was thrombus formation originating at the line of anastomosis.


Clinical evaluation of a microporous membrane oxygenator. 1. The microporous membrane oxygenator has a maximum priming volume of 400 ml. and transfers up to 250 ml. of O2. 2. CO2 transfer is excessive when ventilating the oxygenator with 100% O2. CO2 should be added to the ventilating gas. 3. The oxygenator has performed satisfactorily in 90 cases of cardiac surgery with perfusion time up to nine hours. 4. Precautions are described to prevent possible accumulation of water vapor in the oxygenator.


Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs. Fluid-filled lumina of fetal rat lungs contain lamellar bodies (LBs) as well as tubular myelin (TM), both of which are thought to be stores of phospholipid-rich pulmonary surfactant. The alveolar epithelium is believed to secrete LBs, but neither the origin nor the mechanism of TM formation is entirely certain. The main objective of this study was to determine the relationship between secreted LBs and TM and to define membrane phenomena which occur during TM formation. I examined lung tissues of 20-21 day-old fetuses (day 22 = term) using transmission and high voltage transmission electron microscopy and cytochemistry. My findings indicate that secreted LBs, identified by the presence of an acid-phosphatase reactive core, are the precursor of TM. Secreted LBs are highly organized structures which contain structurally specialized areas, one of which is a "mini-lattice" structure similar to TM. During TM formation, fuzzes or 8.0-nm diameter particles appear on transition membranes, although LB membranes appear to lack both structures. Similar particles are present on TM membranes and are generally associated with membrane intersections. My results provide evidence that TM is formed from LBs within the alveolar lumen by mechanisms which may be complex.


Surgical treatment of acute dissecting aneurysm of the ascending aorta. Since 1959, 51 patients underwent open heart surgery for correction of an acute dissecting aneurysm of the ascending aorta. Upon admission, 33 patients were severely hypotensive or in progressive heart failure. Acute aortic insufficiency was found in 24 patients, and hemiplegia or hemiparesis in four. In 45 patients the ascending aorta was reconstructed with a woven Dacron graft. After excision of the dissected part of the aorta, primary anastomosis or patch aortoplasty was performed in six patients. The aortic valve remained intact in 26 patients, and resuspension of the commissures restored competence of the aortic valve in another nine. Sixteen patients required aortic valve replacement because of disrupture of the commissures. Dissection extended into the coronary ostia in nine cases. Reconstruction of the coronary system was accomplished by reimplantation of the ostia, interposition of a vein graft or aortocoronary bypass. Nine patients died within the early postoperative course from uncontrollable hemorrhage (four), further dissection (three) and myocardial infarction (two). Within the first year after surgery, another five patients died from acute aortic dissection (two), pseudomonas infection causing rupture of the proximal graft anastomosis (one) and myocardial infarction (two). Contraindications of antihypertensive treatment of acute dissection of the ascending aorta are discussed. We recommend prompt surgical intervention in acute dissecting aneurysms of the ascending aorta.


Thermotropic lateral translational motion of intramembrane particles in the inner mitochondrial membrane and its inhibition by artificial peripheral proteins. Freeze fracturing and deep etching have been used to study thermotropic lateral translational motion of intramembrane particles and membrane surface anionic groups in the inner mitochondrial membrane. When the inner membrane is equilibrated at low temperature, the fracture faces of both halves of the membrane reveal a lateral separation between intramembrane particles and particle free, large smooth patches. Such separation is completely reversed through free lateral translational diffusion by reversing the temperature. The low temperature induced, particle-free, smooth membrane patches appear to represent regions of protein-excluding, ordered bilayer lipid which form during thermotropic liquid crystalline to gel state phase transitions. When polycationic ferritin is electrostatically bound to anionic groups exposed at the membrane surface at concentrations which inhibit the activities of cytochrome c oxidase and succinate permease, the bound ferritin migrates with intramembrane particles during the thermotropic lateral separation between the membrane particles and smooth patches. When bound polycationic ferritin is cross-bridged with native ferritin, an artificial peripheral protein lattice forms in association with the surface anionic groups and diminishes the thermotropic lateral translational motion of intramembrane particles in the membrane. These results reveal that the anionic groups of metabolically active integral proteins which are known to be exposed at the surface of the inner mitochondrial membrane migrate with intramembrane particles in the plane of the membrane under conditions which induce lipid-protein lateral separations. In addition, cross-bridging of the anionic groups through an artificial peripheral protein lattice appears to diminish such induced lipid protein lateral separations.


Duodenal erosion by aortorenal dacron graft. Fistulous communications between the bowel and arterial grafts present difficult problems in diagnosis and management. The majority of these cases exhibit retroperitoneal sepsis but in some instances communication between the bowel and the lumen of the arterial graft produces serious hemorrhage. In contrast, the current report describes a patient in whom bleeding occurred twelve years after insertion of an aortorenal graft and the source of bleeding was arterial erosion of an ulcer in the duodenal wall rather than an aortoenteric fistula. Removal of the graft, closure of the ulcer, and nephrectomy were effective in treating the bleeding ulcer and the renovascular hypertension.


Congenital arteriovenous fistula of the internal mammary vessels: report of a case and review of the literature. The case of a congenital arteriovenous fistula of the internal mammary vessels in a 41 year-old woman has been reported, and the pertinent literature briefly reviewed. These rare lesions may become apparent only relatively late in life. The accompanyiny signs are often misleading. In view of the uncertain long-term outlook, surgery seems indicated.


Subcellular localization of a cytochrome P-450-dependent monogenase in vesicles of the higher plant Catharanthus roseus. The intracellular location of a cytochrome P-450-dependent monoterpene hydroxylase from the higher plant, Catharanthus roseus, has been investigated. By differential and sucrose density gradient centrifugation, utilizing marker enzymes and electron microscopy, the monooxygenase was demonstrated to be associated with vesicles having a membrane thickness of 40-60 nm. The vesicles could be distinguished from endoplasmic reticulum, Golgi apparatus, mitochondria, and plasma membrane and were found in light membrane fractions containing provacuoles. Most definitive results were obtained when seedlings were ground in the presence of sand and in a medium containing sorbitol. Upon subjection of the 20,000-g pellet preparation to linear sucrose density gradient centrifugation, a threefold enrichment in hydroxylase activity was afforded in a yellow band having vesicles varying in size from 0.1 to 0.8 mum in diam and having a density of 1.09 to 1.10 g/cm3. Since the monooxygenase has been implicated in indole alkaloid biosynthesis in this plant, the data suggest the compartmentalization of at least a part of this pathway.


Internal carotid artery aneurysm in the neck: management by gradual occlusion of the carotid artery with a Crutchfield clamp. An elderly patient with a large internal carotid artery aneurysm high in the neck is reported. Optimal management by resection and restoration of continuity of the carotid was not felt possible due to the patient's age and the anatomy of the aneurysm. After demonstrating by angiography patency of the contralateral carotid artery and the circle of Willis, the involved carotid was gradually occluded by a Crutchfield clamp. The patient has done well following surgery and the aneurysm has shown progressive decrease in size. This technique is presented to demonstrate an alternate method of management of cervical carotid artery aneurysms when circumstances prevent more definitive treatment.


Binding of microtubules to pituitary secretory granules and secretory granule membranes. Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.


Dispersed mammary gland epithelial cells. I. Isolation and separation procedures. The mammary gland from midpregnant rabbits has been dissociated into individual cells by enzymatic digestion, divalent cation chelation, and gentle shearing. A heterogeneous cell population is obtained, comprising approximately 60% parenchymal cells, approximately 10% myoepithelial cells, and approximately 30% connective tissue cells, including fibroblasts, plasma cells, and microphages. The epithelial cells are characterized by the presence of fat droplets, which in 65% of the cells form large supranuclear vacuoles. Their buoyant density is less than 1.045, allowing their separation from myoepithelial cells and connective tissue cells by isopycnic centrifugation in a density gradient. The homogeneity of the epithelial cell fraction has been assessed by light and electron microscopy. The cells are viable and functionally active as indicated by their ability to exclude vital dyes, incorporate labeled precursors, consume oxygen, maintain intracellular Na+ and K+ concentrations, and retain their structural integrity. In addition, when cultured in Petri dishes, the cells grow as a monolayer, reestablish junctional complexes and retain cell polarity.


Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat-storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.


Distribution of anionic sites on the oviduct ciliary membrane. Polycationic ferritin (PCF) was used as a visual probe for anionic sites on the oviduct ciliary membrane. The binding of PCF to ciliary membranes was dependent on the concentration of the probe in the incubation media. At low concentrations (0.08-0.16 mg/ml), PCF was bound exclusively to the tip of the cilium whereas at higher concentrations (0.32-0.64 mg/ml), ferritin was located at the tip and at the base around the transition region, with occasional scattered clumps on the remainder of the membrane. The base and tip binding was fount to be associated with special surface modifications of the membrane in these regions. At the tip, PCF was bound to a filamentous glycocalyx termed the ciliary crown. Base binding was associated with a system of five to six 140-A high ridges, each of which encircled the membrane of the transition region. The ridges were equally spaced (approxamately 245 A spacing) along the length of the transition region. Since pretreatment of oviduct with either neuraminidase or protease blocked the binding of the probe, the PCF-binding sites appear to be negatively charged glycoproteins or mucopolysaccharides.


Temporal programming of chloroplast and cytoplasmic ribosomal RNA transcription in the synchronous cell cycle of Chlamydomonas reinhardtii. Approximately 90% of the Chlamydomonas reinhardtii chloroplast and cytoplasmic rRNAs was transcribed in the nuclear G1 phase, which occurred during the light period under an alternating light-dark synchronization regime of 12 h each. The remaining 10% of chloroplast and cytoplasmic rRNAs was transcribed from its respective DNAs in the dark period, in the midst of an apparent turnover of a transcription appeared to be prokaryotic in sophistication. The transcription was not interrupted during chloroplast DNA synthesis which occurred during the light period. However, transcription of the nuclear DNA was repressed severely during the nuclear S phase in the dark period. The patterns of incorporation of 32P into chloroplast and cytoplasmic rRNA species in the cell cycle were similar to those of the actual rRNA synthesis as measured optically. However, the quantity of 32P incorporation per unit amount of rRNA synthesized varied considerably during the cell cycle, increasing in all rRNA's during the dark period. 32P incorporation data obtained from continuous and pulse 32P-labeling experiments also revealed a turnover of a small amount of both cytoplasmic and chloroplast rRNAs at the end of the S phase. The 32P incorporation into cytoplasmic and chloroplast rRNAs was well matched temporally with the 32P incorporation into their corresponding ribosomes, indicating that the newly synthesized rRNA molecules are utilized without delay throughout the cell cycle in the assembly of ribosomes.


Membrane changes during cartilage maturation. Increase in 5'-nucleotidase and decrease in adenosine inhibition of adenylate cyclase. To examine the potential participation of the plasma membrane in differentiation, we studied the enzymatic activities of 5'-nucleotidase and adenylate cyclase as a function of chondrocyte maturation. 16-day-old chick embryo tibiae epiphyses were dissected into proliferative, growing and hypertrophying zones. Partially purified membrane fractions prepared by differential centrifugation from the respective tissue segments were assayed for enzymatic activity. Cell suspensions from the same segments were examined cytochemically for the presence of 5'-nucleotidase. The findings show that the 5'-nucleotidase activity of the chick embryo epiphyseal cartilage has the following characteristics: (a) it has a Km of about 25 muM for 5'AMP, and is inhibited by a mixture of 2' and 3'AMP (apparent Ki about 10(-4) M) and by AOPCP; (b) it is predominantly localized at the cell surface but is also detected in the cytoplasm and in association with nuclear heterochromatin; and (c) it increases 10-fold (on a DNA basis) during the maturation of the epiphyseal cartilage cells. The adenylate cyclase activity has these characteristics: (a) it does not change during chondrocyte maturation (on a DNA basis); (b) its susceptibility to adenosine inhibition decreases at least 10-fold. The implication of these findings relative to a possible role of adenosine in cellular communication is discussed.


G2+M arrest of cultured mammalian cells after incorporation of tritium-labeled nucleosides. Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 muC of 3H-methyl-thymidine, 3H-5-uridine, 3H-G-adenosine or 3H-8-adenine. The presence of the 3H-labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 muC 3H-thymidine/ml. The presence of 0.5 muC 3H-thmidine/ml caused a significant increase in the relative proportion of cells in G2+M, even though the population doubling time of the culture appeared to be unaltered.


Electron probe microanalysis of chemical elemental content of single human red cells. Sodium, potassium, iron and sulfur contents of single human red cells were measured using electron microprobe microanalysis. Three preparative procedures were compared, and the most reliable technique was found to be spraying of cells onto polished pyrolytic graphite by atomization. Primary standards were prepared by adjusting the intracellular electrolyte content of red cells, eliminating the need to correct for X-ray absorption. Samples were stable under the electron beam during analysis, and could be stored for long periods of time. Strong correlations were found between the X-ray intensities of iron and sulfur and between potassium and sodium. X-ray intensities of potassium and sodium were found to be directly proportional to internal ionic content. Large populations of single cells could be analyzed and the distribution of their elemental content studied.


Large scale purification and further characterization of a rat liver cell conditioned medium multiplication stimulating activity. The application of roller-bottle cell culture techniques and a relatively simple purification scheme has led to the isolation of milligram quantities of a polypeptide cell multiplication stimulating activity (MSA) from Buffalo rat liver cell conditioned medium. We have characterized the apparently homogeneous MSA with respect to its biological activity, its N-terminal amino acid residue, and its amino acid composition, and have tested the MSA for growth-promoting activity in a number of cell types.


Studies on intercellular adhesion. I. Adhesion of neural retina cells to isotypic aggregates in serum free medium. A quantitative assay for intercellular adhesion, which is a modification of the collecting aggregate assay (Roth et al., '71) is described. The use of mechanically dissociated single cells labeled with 3H-leucine, and of a gyratory shaker increased considerably the efficiency of cell collection. With chick embryo neural retina, it was shown that isotypic cell adhesion occurs in simple synthetic media and even in sodium chloride solution suggesting that divalent cations do not play a major role in adhesion of these cells. Cell collection in media without serum was not affected by metabolic or protein synthesis inhibitors. However, cell adhesion was temperature-dependent since no collection occurred at 4 degrees C but was maximal at 38 degrees C.


Studies on intercellular adhesion. II. Promoting effect of serum and proteins on adhesion of neural retina cells to isotypic aggregates. The quantitative assay described in the preceding paper (Pessac et al., '77) was used to study the effects of serum and various proteins on isotypic adhesion of chick embryo neural retina cells. Fetal bovine, chicken, horse, rabbit and human sera promoted cell adhesion to the same extent. The same sera also enhanced isotypic adhesion of cells from other organs showing that the cell adhesion promoting activity of sera was not organ specific. Neural retina (NR) cell collection in serum supplemented medium was not modified by protein synthesis or metabolic inhibitors and was temperature dependent with a maximum at 38 degrees C. The higher temperature does not seem to be required for repair of the cell surface after dissociation, but for the process of adhesion itself. Various serum fractions and egg albumin showed a cell adhesion promoting activity similar to that of sera.


Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation. Several types of cultured fibroblasts, including chick embryo, human and mouse, were killed by the addition of sodium ascorbate at final concentrations of 0.05-0.25 mM to cultures at the time of inoculation or to attached cells. Ascorbate did not affect the attachment of cells to the substratum. The effect on chick embryo fibroblasts was visible by four hours and by six hours almost all cells had swelled and were becoming detached. By 24 hours detached cells had either lysed or become crenated in appearance. Other end-diol reducing agents and also glutathione and cysteine were effective while gulonolactone, a non-reducing analogue of ascorbate, was ineffective. Preincubation of medium containing ascorbate but no cells, conditions which result in degradation of the vitamin, led to loss of toxicity, indicating that a degradation product was not the lethal agent and that a component of the medium was not converted to a lethal substance. The lethal effect of both ascorbate and glutathione was prevented by the addition of catalase to the medium suggesting that H2O2 formed by intracellular reactions and then excreted into the medium was the cytotoxic agent. This conclusion was supported by the findings that 0.05 mM H2O2 added to chick embryo fibroblasts was lethal and that the effect of this compound on cellular morphology was almost identical to that of ascorbate.


Simple method for the quantitative analysis of tetracyclines by direct fluorimetry after thin-layer chromatography on cellulose plates. A thin-layer chromatographic method is described in which tetracyclines are separated on a cellulose layer by development with aqueous solutions of certain salts (magnesium, calcium, barium and zinc chloride). The spots exhibit good fluorescence in UV light, allowing ready detection without the necessity for treatment with any reagents. This fluorescence can be used for the direct photometric determination of the tetracyclines with good accuracy.


Detection reagent for adenine, guanine, uracil, cytosine and their alkylated bases, nucleotides and nucleosides on thin-layer plates. The detection of alkylated bases at the nanogram level is reported. The reaction is based on two steps, first, chlorination of the bases and, secondly, spraying the chlorinated product with o-tolidine solution to give a dark blue color. The detection of nucleotides and nucleosides is also possible. The optimum conditions and the possible mechanism of the reaction are discussed.


Quantitation of patulin pathway metabolites using gas-liquid chromatography. A gas-liquid chromatographic system which allows most of the metabolites of the patulin pathway to be separated and quantitated has been developed. The metabolites are mostly phenols, and after conversion to their trimethylsilyl derivatives they are efficiently separated on a 3.18 mm O.D. stainless-stell column of 10% QF-1 on Gas-Chrom Q using a temperature programme. The detection limits for the three phenolic acids, 6-methylsalicylic acid, m-hydroxybenzoic acid and gentisic acid are all markedly lower than those obtained in previous systems. The usefulness of the system has been assessed by quantitating the patulin pathway metabolites present in culture filtrates of Penicillium urticae NRRL 2159A.


Determination of N-nitrosoproline at the nanogram level. Two sensitive detection systems are described for the quantitative determination of a nonvolatile nitrosamine, nitrosoproline. One procedure involves denitrosation followed by derivatization of amino product, proline, with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The highly fluorescent NBD-proline compound formed is then identified and quantitated by either thin-layer chromatography or high-pressure liquid chromatography (HPLC). In the second system, the volatile methyl ester of the intact nitrosoproline is prepared, then detected by gas-liquid chromatography (GLC), and confirmed by combined gas-liquid chromatography and mass spectrometry (GLC-MS). Both methods permit the quantitative detection of less than 10 ng of nitrosoproline. However, the HPLC fluorescence technique is approximately ten times as sensitive as the GLC method.


Assay of human plasma for nortriptyline by radioacetylation and thin-layer chromatography. A highly specific and sensitive thin-layer chromatographic method for determining nortriptyline levels in plasma is presented. The procedure involves extracting nortriptyline, acetylating it with radioactive acetic anhydride, resolving acetylnortriptyline by thin-layer chromatography, and measuring its radioactivity.


Polymerization of penicillins. IV. Separation, isolation and characterization of ampicillin polymers formed in aqueous solution. Polymeric substances formed from ampicillin sodium in aqueous solution have been separated according to charge by anion-exchange chromatography on a column of DEAE-Sephadex A-25 using a linear sodium chloride gradient at a constant pH of 7.4. The separated materials, ranging in size from the monomer to the octamer, have been characterized by various functional-group analyses. A homogeneous well-defined ampicillin dimer, tetramer and hexamer have been isolated from the effluent in high yields. The separation and isolation of the ampicillin polymers permit a conclusive establishment of their structures, which in turn has confirmed previous suggestions that the polymers are formed through a chain process involving aminolysis of ampicillin at the beta-lactam carbonyl moiety by the amino group of the side-chain of a second ampicillin molecule.


[Analysis of benzodiazepines and their hydrolysis products, benzophenones, by reversed-phase high-performance liquid chromatography and its application to biological material (author's transl)]. Conditions for the routine separation of some benzodiazepines and their hydrolysis products, benzophenones, by reversed-phase high-performance liquid chromatography have been developed. They were applied to the qualitative and quantitative determination of benzodiazepines and benzophenones in biological material, e.g., blood and urine.


Determination of hydrochlorothiazide in serum by high-pressure liquid chromatography. A quantitative high-pressure liquid chromatographic method has been developed for  the analysis of hydrochlorothiazide in serum in therapeutical concentrations. The method is based on gel filtration of the sera on Sephadex G-15, extraction of the protein-free fraction of the effluent with ethyl acetate and injection of a methanol solution of the drug extract on a reversed-phase column packed with Spherisorb ODS (particle size, 10 mum). The mobile phase is 15% methanol in water. The detection limit is 50 ng/ml of serum. Serum samples from patients receiving hydrochlorothiazide have been analysed by the described method at different hours postdose.


Combined thin-layer and gas-liquid chromatographic identification of tricyclic antidepressants in urine. A combined thin-layer and gas-liquid chromatographic (TLC-GLC) procedure for determination of tricyclic antidepressant drugs in urine is described. GLC on 1.5% OV-17 was utilized to analyzed basic urine extracts directly for the tricyclics and to confirm the identity of spots extracted from thin-layer chromatograms, and TLC was used to confirm the results of these GLC screens. Lower limits of sensitivity for the thin-layer spot extraction procedure were found to range from 2 mug/ml for amitriptyline and imipramine to 10 mug/ml for desipramine and nortriptyline. Turn-around-times for the GLC tricyclic screens were usually less than 1 h. The finding that nortriptyline and desipramine were chemically altered under various extraction conditions was used as a means of confirming the identity of these drugs, and the identity of these chemically altered derivatives was discussed with reference to their low resolution mass spectra.


Determination of chlorinated pesticides in potable water. A procedure is described for monitoring organochlorine pesticides in potable water. Material adsorbed from water on to Amberlite XAD-2 macroreticular resin was eluted with n-hexane and the concentrated eluate was analyzed without further treatment. Electron capture (63Ni) gas chromatography utilizing a column of 4% QF-1 and 2% SE-30 on 80-100 mesh HP Chromosorb W at 180 degrees permitted identification of organochlorine pesticides and indicated the presence of phthalates. Confirmatory qualitative and quantitative analyses were done by specific ion-monitoring mass spectrometry. Levels determined in Ottawa drinking water were 17 ppt alpha-BHC, 1.3 ppt lindane, 0.7 ppt aldrin, and less than 0.05 ppt for each of heptachlor, heptachlor epoxide, dieldrin, endrin, chlordane, o,p'-DDT, p,p'-DDD, p,p'-DDT, and p,p'-DDE.


Gas chromatographic determination of propoxyphene and norpropoxyphene in plasma. Propoxyphene and its major metabolite norpropoxyphene have been determined simultaneously by using gas chromatography. In order to avoid the on-column decomposition of the propoxyphene, derivatives were formed with the aid of lithium aluminium hydride, which cleaves the propionic acid ester of propoxyphene and reduces the norpropoxyphene amide. Promethazine was used as an internal standard. Propoxyphene and norpropoxyphene levels in plasma were determined in samples from six male volunteers receiving a single oral dose of 150 mg of propoxyphene hydrochloride in a sustained-release form (Abalgin Retard).


Improved chromatographic identification of coloured amino acid thiohydantoins. A new N-terminal reagent for peptides and proteins, 4-N,N-dimethylamino-naphthylazobenzene-4'-isothiocyanate, is described which gives purple thiohydantoin derivatives; chromatographic separation of 24 amino acid thiohydantoins is reported. Such standard purple derivatives can be used as markers in the separation of the red 4-N,N-dimethylaminoazobenzene-4'-thiohydantoins. Conversely, standard red thiohydantoins can be used as markers in the separation of unknown purple amino acid thiohydantoins. In two-dimensional thin-layer chromatography, the precision of identifying the unknowns can be greatly improved by running markers with a colour different from the unknowns on the same side of the sheet.


Erythromycin series. V. Quantitative analysis of cladinose and methylcladinoside by densitometry of thin-layer chromatograms. A direct, quantitative, thin-layer chromatographic method is described for the determination of sugar cladinose and methylcladinoside in the presence of other acid-degradation products of the antibiotics erythromycin oxime and erythromycyl-amine. Cladinose and methylcladinoside are separated from compounds which cause interference on pre-coated silica gel F254 plates, and are measured directly on the thin-layer plate using a densitometer. Standard graphs obtained for cladinose and methylcladinoside show a linear relation between the square root of the peak area and the logarithm of the amount of substance present in the spot, as well as between the square of the area and the logarithm of the amount. This method is very successful in stability studies on the antibiotics erythromycin oxime and erythromycylamine in an acid medium. The technique seems to be particularly useful in instances in which the usual analytical methods either cannot be applied or can be applied only with difficulty.


An in situ fluorimetric method for the detection and quantitative analysis of fenitrothion, its breakdown products and other amine-generating compounds. A procedure is described for the in situ detection of primary amine-generating pesticides on thin layers of silica gel. The technique is based on the reaction of fluorescamine with primary amines to yield highly fluorescent derivatives. Nitro compounds are first reduced to their corresponding primary amines. Pesticides studied were aminotriazole, fenitrothion, parathion and parathion-methyl. Possible breakdown products of fenitrothion were also included. Detection limits were in the low nanogram range. The method is simple, rapid and very selective and can also distinguish between primary amines and nitro compounds. An example of a typical chromatographic separation is presented and quantitative aspects are discussed.


Study of aldosterone levels in plasma during pregnancy. A method for the estimation of unconjugated aldosterone in the plasma of pregnant women has been developed. The method is suited to routine analysis of a large number of samples, both from pregnant and non-pregnant subjects. An ethyl acetate extract of plasma is oxidized with sodium periodate to destroy interfering lipids and corticosteroids. Aldosterone is quantitatively converted to aldosteroid-gamma-lactone which is estimated by radioimmunoassay. The method has high specificity and an overall recovery of 80 +/- 5%. The method has been used to establish circulating levels of aldosterone during pregnancy and the menstrual cycle. In early pregnancy, levels of aldosterone were 2-3 times higher than in the follicular phase of the menstrual cycle and a further significant rise occurred at the beginning of the third trimester. No correlation could be detected between plasma aldoserone and plasma progesterone, nor between plasma aldosterone and the birth weight of the baby. Plasma aldosterone levels in women who subsequently developed hypertension were no different from those in women who remained normal throughout pregnancy.


Preferential renal excretion of iodide derived from thyroxine and triiodothyronine deiodination in man. Tracer doses of 131I- (Carrier free), 131I-T3 and 131-T4 were administered po to 19 healthy male volunteers at intervals 2 to 8 weeks to study whether or not part of the iodide generated in the kidney from T3 and T4 deiodination may enter the renal tubular lumen and be excreted in the urine without entering the blood stream. U(urine)/T(thyroid) ratios of the radioactivity from these materials were employed as the index of the comparison. U/T ratios were severalfold higher 24 h after 131I-T3 or 131I-T4 administration than after 131I-. The data indicate that the 131I- derived from T3 and T4 metabolism is more readily excreted into urine than 131I- which reaches the kidney as inorganic iodide.


Immunoreactive luteinizing hormone, follicle stimulating hormone and their subunits in human urine following gel filtration. Urine collected from postmenopausal women, normal men, women and children was prepared by vacuum dialysis and kaolin extraction prior to chromatography on Sephadex G-100. Specific radioimmunoassays for LH, alpha subunit, LHbeta, FSH and FSHbeta were utilized to define the elution patterns of the urinary filtrates, extracts, 2nd IRP-HMG and postmenopausal serum. The following findings were obtained: 1) FSH eluted as a distinct immunoreactive peak whereas LH eluted more diffusely, 2) free alpha subunit was present in all urinary preparations, the 2nd IRP-HMG and postmenopausal serum, 3) free FSHbeta but not LHbeta was detectable in postmenopausal urine, and 4) an LHbeta immunoreactive fragment with an approximate molecular weight of 5000 daltons was present in all urinary preparations and the 2nd IRP-HMG but not in postmenopausal serum.


Successful immunosuppressive therapy in insulin resistant diabetes caused by anti-insulin receptor autoantibodies. A 45-year-old, non-obese female patient with no previous history of insulin administration was found to have extreme insulin resistance and abnormally high plasma immunoreactive insulin in the absence of anti-insulin antibodies in the serum. Clinically, there was no ketonuria. The patient also had evidence of Sjogren's syndrome with several immunologic features including hypergammaglobulinemia, positive antinuclear antibodies, accelerated erythrocyte sedimentation rate and leukopenia. Plasma pancreatic glucagon and C-peptide were elevated, but other endocrinologic abnormalties were not present. In this patient the insulin resistance appeared to be due to anti-insulin receptor antibodies which could be detected even in 1:500 dilution of serum. Immunosuppressive therapy with prednisolone and cyclophosphamide resulted in a decreased level of serum gamma globulin and a concomitant decrease of blood glucose level. After immunosuppressive therapy for eight months, the diabetic syndrome disappeared completely and anti-receptor antibodies in the serum were no longer detectable. Furthermore, insulin sensitivity returned to normal. However, the patient's glucose tolerance deteriorated after the temporary termination of cyclophosphamide treatment and the lowering of prednisolone dosage.


Demonstration of a specific serum carrier protein of nonsuppressible insulin-like activity in vivo. The fate of nonsuppressible insulin-like activity (NSILA-S) was studied by injecting a tracer of 125I-NSILA-S iv into rats. Ten minutes after injection of 125I-NSILA-S alone, 20% of the label is found in serum, whereas after the injection of 125-I-insulin or 125I-NSILA-S together with an excess of cold NSILA-S only 8% of the label are recovered. Sephadex G-200 chromatography at neutral pH of serum after injection of 125I-NSILA-S reveals 2 peaks of radioactivity in the high molecular weight region at 67 and 47% bed volume. Five minutes after injection the peak at 67% starts to disappear, whereas the one at 47% persists with a half-life of 3 h. The latter peak was submitted to Sephadex G-200 chromatography at acidic pH which dissociates NSILA-S from its binding protein. The labeled material obtained by this procedure still exhibits the same binding characteristics to chick embryo fibroblasts as standard 125 I-NSILA-S. A third peak at 90% bed volume corresponding to low molecular NSILA-S is no longer detectable 20 min after injection. A fourth peak at 100% bed volume becomes apparent after 1 h. The half-life and chromatographic pattern of iv injected 125 I-NSILA-S are not changed by the simultaneous administration of insulin or growth hormone. These findings confirm the existence of a specific serum carrier protein for NSILA-S and may explain why endogenous NSILA-S does not exert insulin-like effects under physiological conditions in vivo.


Cortisol excretion levels and daytime sleep in one-year-old infants. Urine was collected from 20 one-year-old infants from the time of awakening in the moring for 8 consecutive h. Free cortisol was measured by competitive protein-binding analysis in each urine sample. The mean cortisol excretion rate for the entire collection period was .228 +/- .019 mug/h/kg. The mean excretion rate of sleep related cortisol (1.86 +/- .28 mug/h) was significantly different from the mean rate for awake periods (2.61 +/- .28 mug/h). The difference could not be attributed to time of day. The results suggest a direct relationship between cortisol production and daytime sleep-wake states in the one-year-old.


Urinary sodium, potassium and aldosterone in Duchenne muscular dystrophy. Four adolescent boys with Duchenne (progressive) muscular dystrophy (DMD) of 10-11 years duration and six normal boys of similar age were studied on a metabolism ward for 22 days. Sodium and potassium intake was as follows: Period I, Na 60 mEq, K 60 mEq; Period II, Na 10, K 60; Period III, Na 10, K 95-150; Period IV, Na 60, K 60. The differences between the DMD group and the group of normal boys for sodium and potassium in serum and urine and for urinary aldosterone were not significant. These findings show that the pathologically elevated sodium-potassium ratio in skeletal muscle of patients with DMD is not due to increased aldosterone or other causes of renal wastage of potassium.


L-Dopa stimulated growth hormone release in the blind. Following L-Dopa administration (single oral dose of 500 mg), plasma Growth hormone (GH) levels significantly rose in 7 out of 8 normal subjects (aged between 64-83) and in 4 out of 8 blind subjects (aged between 60-88). The mean peak +/- SE in the 8 normal subjects was 24.9 +/- 4.9 ng/ml and in the 8 blind subjects was 11.5 +/- 2.8 ng/ml. These findings represent an additional anomaly in pituitary hormone release in the blind and may indicate that dopamine receptors in the hypothalamus are less sensitive to L-Dopa in blind people than in normal subjects.


The effect of dopamine infusion on insulin and glucagon secretion in man. The effect of dopamine on the release of insulin and glucagon was investigated in eight adult subjects. Dopamine infused iv at a rate of 4 mug/kg/min X 2 hr, unaccompanied by cardiovascular changes, induced a prompt and significant increase in plasma insulin and glucagon and in plasma glucose levels. These findings suggest that dopamine may stimulate both alpha and beta cells.


The effect of osmotic stimuli on prolactin secretion and renal water excretion in normal man and in chronic hyperprolactinemia. An osmoregulatory role for prolactin (PRL) in man has been postulated, and PRL secretion has been reported to be influenced by osmotic stimuli. Clinical observation, however, does not support this notion. The effects of water loading, hypertonic saline infusion and nicotine on serum PRL and on renal water metabolism were investigated in 6 normal subjects and in 8 patients with chronic hyperprolactinemia (four with and four without demonstrable pituitary tumors). None of the patients had thyroid, adrenal or vasopressin deficiency. Renal walter handling in these patients was indistinguishable from normal. Likewise, serum PRL was not affected by the stimuli employed in either the normal subjects or the patients. No correlation between degree or duration of hyperprolactinemia and renal water metabolism was found. It is concluded that PRL is not an important osmoregulatory hormone in man.


Decreased binding of insulin by receptors on placental membranes from diabetic mothers. Specific binding of insulin to microsomal membranes from the placentae of insulin-dependent diabetics was significantly decreased when compared with normals. This was due to an apparent decrease in the concentration of insulin receptors.


Cushing's disease: growth hormone response to hypoglycemia after correction of hypercortisolism. Growth hormone responses to insulin-induced hypoglycemia were studied in 17 patients with inactive Cushing's disease. A normal GH rise was found in 9 of 9 patients without evidence of progressive pituitary tumor after bilateral adrenalectomy and 3 of 4 patients after correction of hypercortisolism by transsphenoidal removal of pituitary microadenomas. In contrast, 3 of 4 patients with Nelson's syndrome had impaired GH responsiveness to hypoglycemia. These results show that GH responsiveness is normal in the majority of patients with inactive Cushing's disease and do not support the concept of a primary hypothalamic or central nervous system abnormality of GH regulation in Cushing's disease.


Serum medroxyprogesterone acetate (MPA) concentrations and ovarian function following intramuscular injection of depo-MPA. A sensitive radioimmunoassay measuring serum medroxyprogesterone acetate (MPA) has been developed in order to measure and correlate serum MPA concentrations and ovarian function in women following im administration of deop-MPA (DMPA), employing goat anti-MPA-3-(O-carboxymethyl) oxime-bovine serum albumin and MPA-3-(O-carboxymethyl) imino-125I-iodohistamine. In the 3 women studied, im injection of 150 mg of DMPA yielded brief initial serum MPA concentrations ranging from 1.5 to 3 ng/ml for a few days. Serum MPA concentrations gradually declined and remained relatively constant at about 1 ng/ml for 2 to 3 months, declined gradually thereafter reaching 0.2 ng/ml during the 6th month and became undetectable (less than 0.02 ng/ml) about 7-1/2 to 9 months following administration. Serum estradiol remained at early to midfollicular phase levels for 4 to 6 months after DMPA injection and rose to preovulatory levels when serum MPA levels fell below 0.5 to 0.25 ng/ml. Ovulation, however, as evidenced by serum progesterone concentrations did not occur, apparently due to suppression of the LH peak by positive feedback inhibition. Prolonged inhibition of cyclic ovarian function following DMPA injection is caused by slow MPA absorption and persists until serum MPA levels have decreased below 0.1 ng/ml or become undetectable about 7 to 9 months after DMPA administration.


The effect of posture and saline loading on plasma renin activity and aldosterone concentration in pregnant, non-pregnant and estrogen-treated women. The effect of contraceptive ingestion and pregnancy on components of the renin-angiotensin-aldosterone system was compared in 17 non-pregnant women, 8 non-pregnant women taking oral contraceptives, and 11 pregnant women. Plasma renin substrate concentrations and the dynamic responses of plasma renin activity and aldosterone concentrations to upright posture and iv saline infusion were evaluated. Renin substrate was significantly higher in those women taking oral contraceptives and among the pregnant subjects than the other non-pregnant group. No significant differences in plasma renin activity or aldosterone concentrations were seen before or after postural stimulation among the 2 non-pregnant groups. After saline loading renin was higher and aldosterone lower in the contraceptive-treated group. In contrast, the pregnant group had significantly higher values at every point. The response to posture and saline among the pregnant subjects was similar in direction and magnitude to those of the non-pregnant groups. The urinary excretion of sodium before and after saline infusion was significantly lower in the pregnant group than in the non-pregnant groups. These observations suggest that estrogen-induced increases in renin substrate do not alone account for the increases in the renin-angiotensin-aldosterone system observed during pregnancy, but rather such increases appear to represent a physiological response to increased sodium need during pregnancy.


Wall-less microbial isolate from a human renal biopsy. An orgainsm was isolated from a kidney biopsy of a patient with renal failure. Electron microscopy revealed an ultrastructure very similar to that of a bacterial L-form or mycoplasma. But macroscopically, its colonial morphology was unusual and distinct from that ascribed to these wall-less organisms. This isolate lacked a rigid cell wall and required a hypertonic medium with serum for growth. Also, a long incubation period was essential for its growth, and use of hand lens was necessary for detection on solid medium.


Media-dependent oxidase reaction in a strain of Aeromonas hydrophila. A strain of Aeromonas hydrophila isolated from bile was oxidase positive only when grown on nonselective media. The isolate was oxidase negative when grown on gram-negative selective and/or differential media. It is proposed that oxidase tests on gram-negative rods be performed on colonies grown on nonselective media to assure valid results.


Numbers and types of anaerobic bacteria isolated from clinical specimens since 1960. Between 1960 and 1974, 826 specimens, excluding stool, urine, sputum, and blood, yielded 689 (83%) positive cultures, of which 403 (58.5%) contained anaerobic bacteria. This represents 48.8% of the total specimens cultured. Isolates from 153 specimens obtained and stocked from 1965 to 1974 were reidentified by current criteria. Gram-negative bacilli, primarily bacteroides, were the most frequently isolated anaerobes, being found in 70% of 153 anaerobe-positive specimens and accounting for 42% of the total anaerobes isolated. Gram-positive cocci were second in occurrence, being found in 66% of 153 specimens and accounting for 40% of the total isolates. Bacteroides fragilis was by far the most frequently isolated species. Compairson of 14 years of cumulative data with data from current studies covering 1- to 2-year periods indicated that the anaerobes isolated from clinical material have not changed significantly in type or relative numbers.


Rheumatoid factor: a cause of fals positive histoplasmin latex agglutination. Ten of thirteen patients with positive histolatex agglutination titers of 1:32 or greater had no evidence of acute histoplasmosis. Three of these false positives had rheumatoid arthritis. A fourth had a rising mycoplasma complement fixation titer, and the fifth had a high titer of cold agglutinins. All of these are associated with abnormal immunoglobulin M production. To evaluate the role of rheumatoid factor in producing false positive histolatex agglutination, the histolatex test was performed on sera from 32 patients having rheumatoid factor at a titer of 1:40 or greater. Four of these sera agglutinated the histoplasmin-coated latex particles at titers of 1:32 or greater. Review of clinical records suggests the this reactivity is nonspecific. It is our purpose to call attention to rheumatoic factor as a cause of false positive histolatex agglutination.


Dermatophyte isolation media: quantitative appraisal using skin scales infected with Trichophyton mentagrophytes and Trichophyton rubrum. Various dermatophyte isolation media were quantitatively appraised by colony counts for their ability to support growth when suspensions of trypsin-treated infected-skin scales were used as inocula. Evaluation included guinea pig skin scales infected with a granular Trichophyton mentagrophyton mentagrophytes, and one trial was conducted using human skin scales infected with Trichophyton rubrum. No differences in the ability to support growth were observed among Taplin Dermatophyte Test medium, Dermatophyte Test Medium (Schering Diagnostics, Port Reading, N.J.),Sabouraud medium plus cycloheximide and chloramphenicol, Youssef medium for dermatophytes product no. 103S semisynthetic lots no. 106 and no. 107. With skin scales not grossly contaminated with bacteria, similar counts were obtained with Littman oxgall agar without antibiotics. Youssef medium for dermatophytes product no. 103N natural lot no. 109 was significantly different from the other media in that lower counts were obtained.


Cardiobacterium hominis endocarditis: description of two patients and characterization of the organism. Two cases of endocarditis caused by Cardiobacterium hominis are reported. In both instances infection was subacute and characterized by (i) implantation on abnormal valves, (ii) chronic course lasting weeks to months before recognition, and (iii) rapid clinical and bacteriological response to penicillin, as well as other antibiotics commonly used to treat infections caused by gram-negative bacilli. Our isolates of C. hominis are compared with strains in the National Institutes of Health culture collection. Optimal growth requires yeast extract and incubation at 37 degrees C with increased humidity and supplemental CO2. The production of indole, a positive oxidase reaction, and characteristic sugar fermentation distinguish C. hominis from other slow-growing, gram-negative bacilli.


Origin of acetyl strophanthidin-induced ventricular arrhythmias. To examine the origin of digitalis-induced ventricular tachycardia (VT), acetyl strophanthidin (AS) (25 mug/min) was perfused into a limited zone of myocardium in intact anesthetized dogs through a catheter placed fluoroscopically in the left anterior descending artery without ischemia. A second catheter in the great cardiac vein sampled venous effluent from this region. His and left bundle branch depolarizations were recorded and bipolar intramural electrograms from endocardial and epicardial sites within the anterior descending region were obtained. No conduction alterations preceded arrhythmia. Cardiac venous K+ rose from 3.3 +/- to 4.4 +/- 0.2 meq/liter (P less than 0.001), indicating egress from the perfused zone. 10 animals (Group 1) were sacrificed 2 min after onset of VT while 11 (Group 2) continued until fibrillation (4-14 min). All showed normal (endocardial leads to epicardial) transmural depolarization during sinus rhythm, but 10/21 demonstrated reversal, usually late during VT, including 8/11 in Group 2. Epicardial activation preceded fascicular activation and QRS. Recordings from the border and circumflex regions in 10 additional dogs (Group 3) demonstrated activation reversal only in the border zone. Myocardial K+ was reduced (mean 63 +/- 1 mueq/g) and Na+ increased (mean 41 +/- 2 mueq/g) in the perfused zone (nonperfused circumflex area K+ 72 +/- 1, Na+ 33 +/- 1 mueq/g, P less than 0.001 for both); changes were similar in inner and outer ventricular wall. In related experiments, subepicardial injections of AS induced activation reversal within the immediate area, similar to recordings during coronary infusion. Reversed transmural activation with early epicardial depolarization suggest VT arises within myocardium; electrolyte gradients between adjacent regions may be causative.


Ventilation-perfusion inequality in chronic obstructive pulmonary disease. A multiple inert gas elimination method was used to study the mechanism of impaired gas exchange in 23 patients with advanced chronic obstructive pulmonary disease (COPD). Three patterns of ventilation-perfusion (Va/Q) inequality were found: (a) A pattern with considerable regions of high (greater than 3) VA/Q, none of low (less than 0.1) VA/Q, and essentially no shunt. Almost all patients with type A COPD showed this pattern, and it was also seen in some patients with type B. (b) A pattern with large amounts of low but almost none of high VA/Q, and essentially no shunt. This pattern was found in 4 of 12 type B patients and 1 of type A. (c) A pattern with both low and high VA/Q areas was found in the remaining 6 patients. Distributions with high VA/Q areas occurred mostly in patients with greatly increased compliance and may represent loss of blood-glow due to alveolar wall destruction. Similarly, well-defined modes of low VA/Q areas were seen mostly in patients with severe cough and sputum and may be due to reduced ventilation secondary to mechanical airways obstruction or distortion. There was little change in the VA/Q distributions on exercise or on breathing 100% O2. The observed patterns of VA/Q inequality and  shunt accounted for all of the hypoxemia at rest and during exercise. There was therefore no evidence for hypoxemia caused by diffusion impairment. Patients with similar arterial blood gases often had dissimilar VA/Q patterns. As a consequence the pattern of VA/Q inequality could not necessarily be inferred from the arterial PO2 and PCO2.