Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine.

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.


Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation.

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.


Ontogeny of human neutrophil granulocyte alkaline phosphatase.

Human embryonic neutrophils (N) in the liver (from 8.5 mm crown-rump length) are alkaline phosphatase (AP) negative during the first trimester of pregnancy. Early bone marrow granulocytes (from eleventh to sixteenth weeks of gestation) behave similarly. Only a small percentage of slightly AP positive cells could be found. Occasional cells with strong NAP reaction appear in the second trimester. NAP positivity greatly increases in the third trimester and term-babies have a somewhat higher than normal NAP activity in circulating blood. Unlike NAP reaction, naphthol-AS-D-chloroacetate esterase and peroxidase reactions are positive even in the earliest (AP negative) neutrophils.


Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates.

Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...


The ATP-dependent concentration of calcium by a Golgi apparatus-rich fraction isolated from rat liver.

Fractions rich in Golgi apparatus were isolated from female rat liver and incubated with different concentrations of MgATP and calcium. Calcium was sequestered by the fraction in the presence but not in the absence of ATP. The kinetics of the uptake process showed an exponential type accumulation to a maximum value which could be explained by a 'pumpleak' mechanism where Vmax was estimated at 6.7 nmol calcium/mg protein/min. Apparent Km for ATP was 1 mM and for calcium was 85 micron. The uptake is not inhibited in 5 mM azide, nor is it enhanced in 5 mM oxalate, suggesting that the sequestration is not caused by contamination from mitochondria or microsomes. Parallel experiments on a fraction rich in plasma membrane show at least a 10-fold reduction of activity over the Golgi apparatus-rich fraction, which is interpreted as evidence that plasma membrane contamination in the Golgi apparatus-rich fraction is not primarily responsible for the calcium sequestration activity. Morphometric analysis showed that about 85% of the recognizable membrane profiles in the fraction was associated with elements of the Golgi apparatus. Further evidence is given which suggests that at least 90% of the fraction was uniform in its ability to sequester calcium. In preliminary experiments it was not found possible to separate galactosyltransferase activity (a secific enzyme associated with the Golgi apparatus) from the calcium-sequestration mechanism. The stoichiometry of the hydrolysis of ATP associated with the uptake of calcium is the same as that shown by other isolated membrane systems.


Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes.

The process of infection of lupin nodule cells by rhizobia was examined using thin-section and freeze-fracture electron-microscopic techniques to characterize the properties of different membranes and to establish relationships between them. The membranes of the Golgi bodies and the endoplasmic reticulum stained with zinc iodide-osmium tetroxide but not with phosphotungstic acid or silver. By contrast the infection thread membranes, peribacteroid membranes, plasma membranes and membranes of cytoplasmic vesicles did not stain with zinc iodide-osmium tetroxide but stained with phosphotungstic acid and silver. The peribacteroid membranes and plasma membranes are, however, different from each other since the particle density on the E face of freeze-fracture replicas of plasma membranes was twice that on the E face of the peribacteroid membranes. An examination of the tips of the infection threads in the cytoplasm of the plant cells, showed that the rhizobia bud off from the infection threads enclosed in the infection thread membranes. The rhizobia continue to divide still surrounded by membranes of plant origin, namely the peribacteroid membranes. Cytoplasmic vesicles are observed in both thin-section and freeze-fracture preparations of nodule tissue closely associated with, and apparently produced by, Golgi bodies. Formation of the walls and membranes of the infection threads and of the peribacteroid membranes involves fusion of the cytoplasmic vesicles with these membranes. It is proposed that the process of infection of plant cells in lupin nodules involves a change in the function of the Golgi body system for the biogenesis of plant cell walls and plasma membranes to include the synthesis of the walls and membranes of the infection threads and also the peribacteroid membranes.


Genetic analysis of developmental mechanisms in Hydra. IV. Characterization of a nematocyst-deficient strain.

The authors have previously found that mutant hydra strains showing various types of developmental defects can be isolated through sexual inbreeding of wild hydra. One such defective strain, called nem-4, contains virtually no stenoteles, one of the four types of nematocysts present in hydra, in its tentacles. However, stenoteles are present at a normal level in the body column of this strain, and they are turned over also at a normal rate. Grafting experiments between the head region of nem-4 hydra and the body column of wild type hydra (and vice versa) showed that wild type stenotele nematocytes can move into nem-4 tentacles but that nem-4 stenotele nematocytes can not move into the wild type tentacles. These observations suggest that the stenotele nematocytes are produced normally by differentiation from the interstitial cells in the body column of nem-4 hydra, but that they are somehow prevented from migrating into the tentacles in this strain.


Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules.

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1-153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.


The maintenance of kappa during macronuclear reorganization in Paramecium tetraurelia.

Kappa particles were injected into stock-51 sensitives (KK) of Paramecium tetraurelia to investigate the maintenance of kappa by gene K during macronuclear reorganization. Injection into exconjugants and autogamous cells followed by macronuclear regeneration, induced by microsurgical removal of macronuclear Anlagen, resulted in maintenance of kappa particles by a considerable proportion of the clones descended from the recipient paramecia. Injection into animals that underwent normal reorganization however usually failed to yield particle-maintaining paramecia.


Ultrastructural localization of cationic proteins in human polymorphonuclear leukocytes.

The present investigation is concerned with the use of the post-formalin ammoniacal silver reaction to detect the arginine-rich cationic proteins in human polymorphonuclear leukocytes at the ultrastructural level. These proteins appear to function as neutral proteases in antibacterial action and as mediators of inflammation. Originally, the ammoniacal silver reaction relied upon primary fixation in dilute formalin which prevented optimum fixation of tissues. This study shows that by using the proper sequence of glutaraldehyde fixation and the ammoniacal silver solution in conjunction with osmium tetroxide treatment, better fixation of the tissue and localization of the ammoniacal silver reaction can be achieved. Also, the ammoniacal silver reaction in human polymorphonuclear leukocytes is exclusively located in the large, crystalline cytoplasmic granules of eosiniphils. All other cytoplasmic granules of neutrophils, eosinophils, and basophils were found to be devoid of the ammoniacal silver reaction product. These results are contrary to previously published experimental data. Possible explanations for this discrepancy are discussed.


Supercoiling of DNA and nuclear conformation during the cell-cycle.

When cells are lysed in solutions containing high concentrations of salt and a non-ionic detergent, structures are released which retain many of the morphological features of nuclei. These nucleoids contain superhelical DNA but are depleted of nuclear protein. We have analysed DNA conformation in nucleoids derived from HeLa cells synchronized at different stages in the cell cycle. The gross differences in nuclear morphology seen during the cell cycle are reflected in the morphology of the nucleoids; for example, the individual chromosomes of mitotic cells remain identifiable and aggregated within the mitotic nucleoid. The sedimentation rate of nucleoids in sucrose gradients reflects the gross nuclear morphology; the small S-phase nucleoids sediment 9 times faster than the large mitotic nucleoids. Despite these large differences at the gross level of organization, both the degree of supercoiling and the size of the units in which supercoiling is maintained are roughly similar in the nucleoids derived from cells in the different phases. The protein content of the various nucleoids is also very similar. Like the nucleoids made from randomly growing cultures of cells, mitotic nucleoids are excellent templates for the RNA polymerase of Escherichia coli.


Small nuclear RNAs: an association with condensed chromatin in amoebae.

For the purpose of studying the relationship of small nuclear RNAs (snRNAs) to chromatin, we studied the effect of the chromatin-binding agent actinomycin D (AMD) on the nuclei of amoebae. By electron microscopy we noted that when mitotic cells were placed in AMD their chromatin was highly condensed 3 h later. This contrasts strikingly with the situation in untreated cells, in which the chromatin at the same time is largely decondensed. After arranging, by nuclear transplantation of labelled nuclei into unlabelled cytoplasm, for the only labelled molecules in the cell to be 3H-snRNAs, we noted that in such cells similarly treated with AMD the labelled material was clustered in a form quite similar to that of the chromatin in 3-h post-division, AMD-treated cells. In untreated cells the 3H-snRNAs were distributed essentially uniformly throughout the nucleus. These observations, together with earlier findings that snRNAs associate with chromosomes during mitosis, lead us to conclude that snRNAs have a particular affinity for condensed chromatin.


The effect of antrycide of patterns of RNA synthesis in Amoeba discoides.

Antrycide is an aminoquinaldine whose inhibitory action on the growth of Trypanosoma and Crithidia is not fully understood at the cellular level. The growth of Amoeba discoides in concentrations of antrycide between 0.5 and 2 microgram/ml was reduced considerably, while cells failed to divide in 4 microgram/ml. The effects on growth rate were reversible at least up until 7 days in antrycide. In order to assess the action of this synthetic drug on RNA synthesis in amoebae, the pattern of synthesis in normal cells was investigated using polyacrylamide gel electrophoresis. The profile of high molecular weight RNAs observed depended on the length of time in [3H]uridine, and was only fully developed after 66 h, when 5 peaks could be seen. The relative molecular weights of these peaks (I--V) were 2.45, 1.55, 1.13, 0.8 and 0.52 X 10(6) Daltons respectively. Those of 1.55 and 0.8 X 10(6) corresponded to ribosomal RNAs, the identity of the other peaks is unknown. After growth in 2 microgram/ml antrycide for 4 days, no high molecular weight RNA was found. Use of [14C]adenine/[3H]uridine showed that after 17 h in antrycide there was a loss of ribosomal RNA and increased levels of low molecular weight RNAs, due either to lack of synthesis or to degradation of newly synthesized material. Incorporation of [3H]leucine into hot acid-precipitable protein was inhibited in antrycide-treated cells by at least 50%. A possible explanation of the effect of antrycide on A. discoides was the inhibition of mRNA synthesis for ribosomal proteins, leading to degradation of newly synthesized rRNA. Reduced growth would continue on pre-existing ribosomes and previously synthesized long-lived mRNAs.


Cell movements in a confluent monolayer are not caused by gaps: evidence for direct contact inhibition of overlapping.

According to the hypothesis of contact inhibition of movement, cells in a confluent monolayer are restrained from major overlapping by a directional inhibition of locomotion. This explanation of monolayering proposes that contact between 2 cells locally paralyses the locomotory function, preventing movement in the direction that would lead to overlapping. Consequently, a cell in contact on all sides with neighbouring cells should be immobilized. Yet in strictly monolayered cultures of confluent chick liver or mouse 3T3 cells, we have previously observed both translational cell movements and re-shufflings of relative cell positions. The 'confluence' was not perfect, however, and it seemed possible that the movements observed were due to release from contact inhibition by occasional transitory gaps seen to open up between cells. In the present study, detailed gap experiences and cell movements were recorded for 31 cells over a total of 1637 cell-hours. There was no significant correlation between frequency of gaps experienced and the extent of cell movement measured as neighbour-exchanges. We conclude that gaps are not a major cause of the movements observed. The hypothesis based on contact inhibition of motion, which attempts to explain monolayering indirectly by imposing a restraint on cell locomotion, cannot explain the substantial cell movements seen in the confluent cell monolayer studied here. To explain contact inhibition of overlapping, the evidence favours a more direct hypothesis which places no restriction on cell movement other than that overlapping be avoided. Such direct avoidance of overlapping could result from differences in the strengths with which cells adhere to one another and to the substratum.


Reaction of surface lamella of moth spermatozoa to vinblastine.

Previous ultrastructural studies appear to indicate that the lacinate appendages (highly elaborated laminar structures which cover the surface of moth spermatozoa) may be intracellular derivatives of transient microtobules found in the elongating spermatids of these insects. Additional support for this theory is supplied by the present study in which testes of the warehouse moth Ephestia cautella were treated in vivo with the antimitotic agent vinblastine sulphate. Solutions containing 10(-5) M vinblastine caused the lacinate appendages to become poorly resolved, and at 10(3) M they disappeared. This concentration-dependent response of the appendages to vinblastine resembles that of tubulin-containing structures.


Formation and growth of gap junctions in mouse myocardium during ontogenesis: a freeze-cleave study.

The freeze-cleave technique demonstrates the presence of gap junctions at early stages of mouse cardiac muscle ontogenesis. The formation and growth of these junctions were studied at 4 stages of development: 10, 14, 18 days post-coitum (dpc) and at the adult stage. The diverse aspects of the gap junctions are interpreted as different steps in their formation. The first indication of this formation seems to be the presence of linear arrays of 9-nm particles on PF faces. At one end of these arrays a small aggregate of particles appears which acts as nucleation site and grows by incorporating individual gap particles and/or linear arrays. Nexuses with arms and/or central particle-free zones would represent intermediate steps in the formation of junctions. The largest nexuses could be formed by fusion of smaller ones and/or by accretion of gap particles. Analysis of the size distribution of gap junctions shows their growth during their development. At 10 dpc the surface area (S) of nexuses ranges from 0.1 to 3 x 10(-2) micrometer2, at 14 dpc from 0.1 to 15 x 10(-2) micrometer2, at 18 dpc from 0.1 to 26 x 10(-2) micrometer2, and at the adult stage from 0.1 to 54 x 10(-2) micrometer2. The percentage of large nexuses (Sgreater than 0.5 x 10(-2) micrometer2) steadily increases from 10 dpc to the adult stage. Fixation by glutaraldehyde before glycerol infiltration does not induce any modification in the size distribution of adult heart gap junctions.


Effects of pronase and concanavalin A upon the freeze-etch morphology of cell membranes of intact human erythrocytes.

Freeze-etch studies reveal that mild pronase treatment with subsequent incubation in concanavalin A induces aggregation of intramembranous particles (IMP) in intact human erythrocytes. This alteration in particle distribution is accompanied by a change in the distribution of the Con A molecules such that they also become clustered on the extracellular etch face. If divalent succinyl Con A is used after pronase instead of tetravalent Con A the IMPs still become clustered. Pronase only, Con A only, or succinyl Con A only does not cause the IMPs to become aggregated. Most surprising is the finding that pronase followed by Con A causes partial haemolysis of the cells whereas pronase only, Con A only, or pronase+succinyl Con A do not cause this haemoglobin loss. These perturbations of the erythrocyte plasma membrane appear to be a result of the pronase+Con A exerting a transmembrane effect on the spectrin. This conclusion is supported by sodium dodecylsulphate polyacrylamide gel electrophoresis of material crosslinked with dimethyl adipimidate dihydrochloride, which indicates that spectrin is more susceptible to being cross-linked after pronase+Con A; i.e. the spectrin is probably aggregated by the enzyme and lectin incubation.


Membrane junctions in the myelin sheath of goldfish lateral nerve.

The myelin sheath of the goldfish lateral line nerve has been studied by means of the freeze-etch technique. Fracture faces of the myelin lamellae reveal linear structural elements which in some respects resemble those of tight junctions. In unfixed myelin rows of particles on the ef face are matched by narrow grooves on the pf face. Fixation with glutaraldehyde produces a partitioning of junctional particles between the 2 fracture faces. These elements are observed most frequently in the outer layers of the sheath where their arrangement is predominantly more or less parallel and longitudinal, merging in places into a more reticulate organization. They also occur at other levels in the myelin and in association with Schmidt-Lantermann clefts. Orientation in these cases appears to be predominantly longitudinal, though circumferentially running elements have been observed. The possible functional significance of these structures and their implications for tissue development, maintenance and growth are discussed.


[Acute pancreatitis. Superficial capsular necrosis and parenchymatous involvement. 115 cases, 11 resections (author's transl)].

The authors report 115 cases of acute pancreatitis who were either operated on or autopsied and emphasise the severity of the glandular parenchymatous involvement which remains, furthermore, difficult to recognise during manual exploration. Necrosis is sometimes limited to the peripheral interstitial tissue and spares the gland itself (the prognosis is then less serious) and has led many surgeons to resections which did not always seem justified a posteriori. The authors refer to pancreatic decapsulation, described by rumanian authors, and suggest a method of operative diagnosis of the extent of glandular necrosis.


[Surgery of retrospinal disinsertion of the posterior cruciate ligament of the knee (author's transl)].

Thus in all cases of an isolated tear of the posterior cruciate ligament, with an interglenoid fragment, whe fixation by a screw or clip was carried out, we obtained good results by simple surgical treatment. In spite of the small size of this series, the results were in perfect harmony with those of other authors, which justifies surgery. In three other cases of a tear of the posterior cruciate ligament, orthopedic treatment was adopted for various reasons; comparison of the results was definitely in favour of surgery owing to the duration of plaster immobilisation and physiotherapy in the first group and the functional sequelae with chronic invaliding laxity in the cases treated conservatively.


[Carcinomas of the transverse colon. A series of 75 cases (author's transl)].

The authors report a series of 75 cases of carcinoma of the transverse colon, involving equally the right and left halves. They rarely arose from the middle segment (10% of the series). Usually the tumour was very large affecting the serous layer with lymph node invasion. Liver metastases were frequent. 50% of patients were aged over 70 years. Theoretically, wide removal attempting to cure the disease means removing both colonic angles and the areas supplied by both the superior and inferior mesenteric arteries. But fairly often the existence of vascular variations in the transverse mesocolon permits one to restrict the extent of the resection whilst saving the basic principles of radical removal. Furthermore, in 20% the treatment was purely palliative. The operative mortality was high (25%). The five year survival, in the whole series was about 50%.


[Late signs of vascular complications of closed trauma of the shoulder (author's transl)].

The authors report four cases of vascular complications of closed injuries of the shoulder, which appeared later, some time after the initial accident. There was one false aneurysm of the axillary artery at the forty sixth day after a fracture of the upper humeral diaphysis with elongation of the brachial plexus; one case of acute ischemia of the upper limb 3 years after a dislocation of the shoulder; one case of thrombosis with gangrene of the hand twelve years after a fracture-dislocation of the head of the humerus and one case of chronic ischemia of the forearm with severe changes in the vessels below one year after a fracture of the clavicle which had caused an aneurysm of the subclavian artery. The clinical, pathological and physiopathological study showed the enormous possibilities of the collateral circulation in the axilla. An arterial lesion may thus remain undiagnosed during the initial trauma. The authors emphasise the importance of the initial examination with a search for disappearance of the peripheral pulses. In the light of these four cases, it seems necessary to explore as a routine any arterial lesion complicating a shoulder injury.


[Vascular complications of exostosis. About an exceptional case of true femoral aneurysm (author's transl)].

An exceptional observation of a true femoral aneurysm complicating an exostosis is related. From a review of 60 cases of the literature, the authors study the different vascular complications induced by osteochondroma. In 3 cases out of 4, the vascular injury consist in a false aneurysm; in 1 case out of 5 vascular compression is the point in question; only 4 cases of true aneurysm have been collected. After study of the different clinical aspects of the disease, the authors emphasize the need of surgical treatment associating removal of exostosis and restoring vascular axis. They suggest preventive removal of exostosis situated on a vascular axis to hinder the onset of such accidents.


Chromatographic profile of high boiling point organic acids in human urine.

The profile of high boiling point organic acids in urine samples from both normal subjects and patients suspected of having some form of metabolic disorder has been determined by combined gas chromatography-mass spectrometry. Fifteen different compounds eluting after hippuric acid have been identified, including two, cinnamoylglycine and acetyltributylcitrate, which have not been recognised previously. Relative retention times and abbreviated mass spectra of the identified compounds are presented.


O-trimethylsilylquinoxalinol derivatives of aromatic alpha-keto acids. Mass spectra and quantitative gas chromatography.

As an extension of earlier work on aliphatic alpha-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic alpha-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilylation. The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0 degrees. The final derivatives are stable for weeks at room temperature. Low resolution mass spectra are reported. The fragmentation mechanisms are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinois, O-(TMS-d9)-quinoxalinois and O-TMS-6(7)-chloroquinoxalinois.


Liquid chromatographic analysis of sebum lipids and other lipids of medical interest.

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography-mass spectrometry, is very easy. Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.


Microchromatography of hemoglobins. VIII. A general qualitative and quantitative method in plastic drinking straws and the quantitative analysis of Hb-F.

The microchromatographic procedure for the quantitative analysis of the hemoglobin components in a hemolysate uses columns of DEAE-cellulose in a plastic drinking straw with a glycine-KCN-NaCl developer. Not only may the method be used for the quantitative analysis of Hb-F but also for the analysis of the varied components in mixtures of hemoglobins.


Sensitive fluorimetric method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography and its application to human blood.

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles. The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 +/- 3.1, and spermine 8.4 +/- 2.8 nmoles/ml packed erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml. The polyamine content of the erythrocytes from patients with malignant neoplasms was significantly elevated.


High-performance liquid chromatographic determination of hippuric acid in human urine.

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.


A radioimmuno-chromatographic scanning method for the analysis of testosterone conjugates in urine and serum.

A method for the analysis of testosterone (and 5alpha-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were grought to pH 1 and extracted with a diethyl ether-methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological mateiral.


Isoelectric focusing in polyacrylamide gel carried out with a simple device for power regulation. Application to mammalian growth hormones.

A unit is described to regulate the electrical power delivered to an isoelectric focusing cell. The device consists in a resistance set, in series with a power supply, which can be adjusted to a suitable resistance value in order to permit only minor changes in wattage. Simultanoeus monitoring of voltage and current is also carried out with a pen-recorder adaptor. The improvements in operative conditions achieved by these means are tested by the fractionation of mammalian growth hormones.


Microtechniques for the gas chromatographic determination of barbiturates in small blood samples.

A method for the determination of therapeutic levels of barbituric acids in 25 microliter of whole blood is described. After extraction and controlled concentration of the extract to a volume of 5 microliter, the barbituric acids are N,N'-dimethylated using a microrefluxer. Of the total extract 20-100% is injected into the gas chromatograph. Low blanks, recoveries of 70--80% and peak ratios that are comparable to those in calibration experiments are obtained provided the detailed working instructions are followed strictly. In addition, barbiturates were determined (1 ng in 25 microliter blood) using column-switching devices and nitrogen-sensitive detection.


A micro liquid column chromatography procedure for twelve anticonvulsants and some of their metabolites.

Solvent extracts of 50 microliter volumes of serum were sufficient for the determination of twelve anticonvulsant drugs. The liquid chromatography procedure utilized a C-18 reversed-phase column and isocratic elution with 15% acetonitrile in water. No derivatization was required. Eluted anticonvulsants were detected by UV absorption at 195 nm and quantitated by drug--internal standard peak area ratios. The procedure provided linear working curves over the concentration range from 1 to 100 mg/l of drug in the serum. The procedure for serum provided recoveries of the drugs from 92 to 101%. Within-day precision was about 4% and day-to-day precision was about 6.5%. The procedures has been applied to urine samples to facilitate bioavailability studies. Data are also given for several metabolites. There is a discussion of many practical aspects of the procedure to improve the reliability of the results.


Reversed-phase liquid chromatography of adriamycin and daunorubicin and their hydroxyl metabolites adriamycinol and daunorubicinol.

Adriamycin and daunorubicin and their metabolites adriamycinol and daunorubicinol were separated by reversed-phase liquid chromatography using LiChrosorb RP-2, RP-8 and RP-18 as supports and acetone, acetonitrile and alcohols as organic modifiers in the mobile phase. The highest separation selectivity was obtained using a mobile phase containing low concentrations (less than 20%) of acetonitrile. The length of the hydrocarbon chains of the surface-modified silica supports had no significant influence on the selectivity. The lowest capacity factor was obtained with 40-60% of organic solvent in the mobile phase. Increasing the length of the hydrocarbon chains of the supports increased the retention of the solutes.


Rapid reversed-phase high-performance liquid chromatographic analysis of steroid products.

The optimisation of conditions for the application of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of steroid products is discussed. The influence of temperature and flow-rate on column efficiency are described. At 60 degrees, the reduced velocity required to give maximum efficiency, is approximately twice that found at room temperature, allowing faster through-put. (hv) curves are reported for 5-micron Spherisorb-ODS and Hypersil-ODS material for corticosteroids at 60 degrees. On-column injection techniques are discussed and data presented, showing that for corticosteroids on-column loop injection can be efficient as septum injection using the techniques described.


Determination of paracetamol and its metabolites in urine by high-performance liquid chromatography using ion-pair systems.

Paracetamol (P) and its four main metabolites, the sulphate (S), glucuronide (G), cysteine (C) and mercapturic acid (M) conjugates, are separated on ODS/TMS silica using a standard eluent, water-methanol-formic acid (86:14:0.1, v/v/v), in the order S, G, P, C, M. On addition of the ion-paring cations dioctylammonium (DOA) and tetrabutylammonium (TBA), the retention of S is vastly increased while those of G and M are substantially increased and that is C is reduced. The excessive retention of S and to a lesser extent that of M and G can be controlled by the addition of a suitable concentration of a co-ion such as nitrate. The order of elution is then G, C, P, M, S, although this can be varied by adjusting the amount of nitrate present. Loading of DOA is slow as it is very strongly adsorbed and typical concentrations in the eluent are below 7 mg/l. Loading by TBA is rapid with typical concentrations being around 200 mg/l. The effects of added co-ions such as nitrate can be explained in terms of simple ion-pair equilibria. Equilibration with respect to added salts is rapid. Application of the technique to analysis of therapeutic and overdose urines shows the presence of at least three additional metabolites, one of which is identified by mass spectrometry as most probably 3-methoxyparacetamol. Another appears to be a methoxymercapturic acid derivative. There is further evidence for a group of metabolites that elute unresolved from overdose urines as a broad band after the main metabolites.


Liquid chromatography in the monitoring of plasma levels of antiarrhythmic drugs.

High-performance liquid chromatography has been employed in the development of assay methods for six antiarrhythmic drugs, disopyramide, lidocain, tocainide, procainamide, aprinidine and quinidine. Liquid-solid chromatography has been used and separation times of about 5 min have usually been sufficient. Owing to the capacity of the liquid chromatographic system, sample preparation has been minimized to a single extraction and direct injection of a considerable part of the extract. The overall time of analysis is very short and the methods are well suited for monitoring of plasma levels of the antirrhythmic drugs and in some instances (procainamide and disopyramide) also for their main metabolites. UV detection at the optimal wavelength has permitted determinations down to 50 pmole (20 ng) in 1 ml of plasma for the amines with high absorbance.


Determination of oral anti-diabetic agents in human body fluids using high-performance liquid chromatography.

Two widely prescribed anti-diabetic agents for which no simple assay method was previously available can now be determined by high-performance liquid chromatography using a UV detection system. The two drugs investigated were tolbutamide (a sulphonylurea) and phenformin (a biguanide). Tolbutamide can be assayed directly, after a single extraction step, on a reversed-phase system, illustrating the simplicity of the technique for carrying out analyses on underivatised drug compared with gas chromatography. Phenformin was not so easily chromatographhed using straightforward partition systems; however, by the choice of a suitable ion-pair agent it was possible to chromatograph the underivatised drug in a relatively simple reversed-phase system.


Photochemical detection in high-performance liquid chromatography and its application to cannabinoid analysis.

A novel technique of on-line photochemical derivatization is described which can enhance considerably both the sensitivity and specificity of detection in high-performance liquid chromatography (HPLC).. Material eluting from the column is irradiated with a high flux of UV light, which may induce a reaction to form fluorescent or highly UV-absorbing products. The irradiated eluent then passes into a suitable detector. The photochemical reactor as a neglible effect on resolution, and reaction is achieved in 1-5 sec. An example of the use of this technique is in the detection of cannabinol CBN), a component of cannabis, which is converted into a highly fluorescent compound on irradiation with UV light. Thus, if a sample containing CBN is chromatographed and the column eluent irradiated, CBN can be detected (as the fluorescent photoproduct) with a sensitivity of less than 1 ng. If the chromatogram is then repeated without UV irradiation, only naturally fluorescent products are detected. A compairson of the two chromatograms allows these to be eliminated and leads to a very high specificity for the method. This approach is being developed as the basis of a rapid, sensitive and specific method for the detection of cannabinoids in body fluids. It is expected, however, that photochemical derivatization will extend the use of HPLC to many substances that cannot be satisfactorily detected at present.


Specific method for determining uric acid in serum using high-performance liquid chromatography and gas chromatography-mass spectropmetry.

A method using a combination of high-performance liquid chromatography and stable-isotope dilution-mass spectrometry is described for the specific quantitation of uric acid in serum. The procedure involves addition of a known amount of [1,3,9-15n]uric acid, as intenral standard, to the serum sample followed by equilibration with the endogenous analyte. After separation from serum proteins, cationic and neutral compounds by anion-exchange chromatography, the purified uric acid is converted into its tetraethyl derivatives. High-performance liquid chromatography is used to isolate the three major isomeric derivatives for measurement of the isotope ratio m/e 280 to m/e 283. This ratio gives the relative abundances of the molecular ions of natural and of labelled tetraethyluric acid, and from it the amount of uric acid in the original serum specimen is determined. Effective separation of tetraethyluric acid isomers can be achieved by adsorption or reversed-phase high-performance liquid chromatography using n-heptane-isopropanol (80:1, v/v) and methanol-water (3:2, v/v), respectively, as solvent systems.


Investigations of anabolic drug abuse in athletics and cattle feed. II. Specific determination of methandienone (Dianabol) in urine in nanogram amounts.

A high-performance liquid chromatographic method for the determination of the free excreted anabolic drug methandienone in pharmacokinetic studies is described. After extraction of the free steroids from urine, separation on reversed-phase columns leads to quantitative determination of this drug down to 5 ng, and to qualitative detection of less than 1 ng amounts. Because the anabolic drugs and their metabolites are eluted later than the other normally excreted constituents this method is also useful for the routine surveillance of anabolic drug abuse in the sports in general.


Determination of cocaine in human urine, plasma and red blood cells by gas-liquid chromatography.

This paper describes a sensitive and reliable method for the determination of cocaine in human urine, plasma and red blood cells. Cocaine is extracted into cyclohexane from the biological materials at slightly alkaline pH, reduced with lithium aluminium hydride, acylated with pentafluoropropionic anhydride and detected by an electron capture detector. When compared with a gas chromatography-mass spectrometry method the results of cocaine determination correlated highly (r = 0.986). When cocaine was given intravenously to volunteer subjects only 0.2-1.4% of the administered dose was excreted as unmetabolized cocaine in the first 9 h after administration. Plasma and red blood cell levels of cocaine were also determined by this method after intravenous administration.


Measurement of benzoylecgonine and cocaine in urine, separation of various cocaine metabolites using reversed-phase high-performance liquid chromatography.

The application of reversed-phase high-performance liquid chromatography to the measurement of benzoylecgonine and cocaine in urine is described. Following a simple extraction and clean-up procedure, chromatography is performed using a column containing an octadecylsilica coated packing, elution with 17% acetonitrile in pH 2.7 phosphate buffer and ultraviolet detection at 200 or 235 nm. The detection limit is ca.0.1 microgram of drug per ml urine, and using the ethyl ester of benzoylecgonine as an internal standard, benzoylecgonine and cocaine are quantified with coefficients of variation of 7.0 and 2.8%, respectively. The procedure has been applied to urines from subjects receiving intranasal cocaine, and compared to the enzyme multiplied immunoassay technique. The chromatography procedure also permits the separation of norcocaine and benzoylnorecgonine.


Simultaneous quantitation of twenty-two amino acids in nanolitre samples of biological fluid.

The technique of thin-layer chromatography and 14C-dansylation has been used for simultaneous measurements of 22 individual amino acids, taurine, serotonin, and gamma-aminobutyric acid in nanolitre samples of glomerular and renal tubule fluid. Standard curves of each amino acid mixture containing all the others showed excellent linearity at amounts ranging from 5.7.10(-13) to 1.45.10(-11) mole (regression coefficients all greater than 0.95). With careful standardization of dansylation conditions, the technique permits highly reproducible measurement of less than 1 pmole of an amino acid.


Quantitative estimation of salicylic acid and its metabolites by thin-layer densitometry.

A rapid thin-layer densitometric method for the quantitative determination of salicylic acid and its metabolites in urine or plasma is described. The method is specific and very sensitive. Nanogram quantities of salicylic acid and its metabolites, both free and conjugated, may be estimated. Known metabolites, as well as the newly described gentisuric acid, were estimated quantitatively in urine from a patient treated with aspirin.


Determination of chlorthalidone in plasma, urine and red blood cells by gas chromatography with nitrogen detection.

A sensitive and selective gas chromatographic method is described for determining the diuretic and antihypertensive drug chlorthalidone in plasma, urine and erythrocytes. Use is made of an alkali flame ionization detector (nitrogen detector), and the chlorthalidone and internal standard are chromatographed as methyl derivatives. Down to 10 ng of drugs in the biological sample can be measured accurately, with a standard deviation of 5%. Because the concentration of chlorthalidone found in erythrocytes is 50-100 times higher than that in plasma, the influence of haemolysis on the plasma concentration has been investigated. In addition, a pharmacokinetic study with human volunteers revealed that the apparent concentration of the drug found in plasma can be much too low (by more than 50%), if the plasma is not separated from the erythrocytes immediately after venipuncture. Precautions to be observed to ensure correct handling of blood samples (so that results for plasma concentrations will be reliable) are stressed. The findings have application in kinetic studies on chlorthalidone.


A rapid analytical method for measuring toluene in biological specimens.

A rapid and accurate method for the direct extraction and quantitation of toluene in blood and tissues has been developed. The technique involved extraction with methanol, selective adsorption onto Tenax, and desorption from the Tenax with heat and injection into a gas chromatograph. Measurement of standards indicated values were linear over a range of tissue concentrations of 1-1,734 microgram per gram of sample. The procedure described in this paper is a rapid, accurate method for measuring volatile hydrocarbon solvent in both blood and solid tissues of experimental animals. This technique should be applicable to basic research including animal studies, as well as to clinical cases involving abuse of or industrial exposure to solvents.


Quantitative analysis of berberine in urine samples by chemical ionization mass fragmentography.

A highly specific and sensitive method has been developed for the quantitative determination of berberine in human urine. In order to carry out the microdetermination of berberine by chemical ionization mass fragmentography, berberine was reduced with sodium borohydride in methanol to tetrahydroberberine and subjected to gas chromatography-mass spectrometry. Berberine concentrations as low as 1 ng/ml urine can be measured by this method, with [2H3]berberine chloride as an internal standard.


Quantitative determination of tiflorex in human fluids using electron-capture detection.

A procedure is described for the determination of tiflorex and its metabolite nortiflorex in biological specimens. The compounds are converted into their trichoroacetyl derivatives, which are separated on a glass column packed with 3% OV-17 on Gas-Chrom Q, and measured with an electron-capture detector. The mechanism was investigated by gas chromatography-mass spectrometry. The method is rapid, sensitive for concentrations of 1 ng/ml and has been used to measure tiflorex and its metabolite in rat plasma after intravenous administration and in human volunteers after administration by the oral route.


Determination of di (2-ethylhexyl) phthalate, mono (2-ethylhexyl) phthalate and phthalic acid by high pressure liquid chromatography.

A method is presented for the determination of di (2-ethylhexyl) phthalate, mono (2-ethylhexyl) phthalate and phthalic acid in anticoagulant solutions using reverse phase high pressure liquid chromatography.


Isolation and identification of Peptococcus saccharolyticus from human skin.

Peptococcus saccharolyticus was a numerically important organism on the forehead and the antecubital fossa of the arm of ca. 20% of the subjects studied. It was best detected by prolonged (4- to 7-day) anaerobic incubation on a suitably enriched medium but also appeared in smaller numbers in aerobic primary cultures after 4 to 7 days. Initial screening was based on growth patterns in shake cultures and on the relative amounts of growth on aerobic and anaerobic streak plates. The organism was nonhemolytic and usually catalase positive. It produced acid anaerobically from glucose, fructose, and glycerol, but not from maltose, and did not produce lactic acid from glucose.


Immune adherence hemagglutination: alternative to complement-fixation serology.

Immune adherence hemagglutination (IAHA) was compared to complement fixation (CF), using standard procedures, for serological testing of human sera with a number of commercially available antigens. The antigens included herpes simplex, measles, cytomegalo-, and influenza (type B) viruses, as well as Mycoplasma pneumoniae and Chlamydia psittaci (Chlamydia group). The IAHA test was found to be as specific as the CF test, but 4 to 20 times as sensitive with all antigens tested. Antigen titers were also higher with the IAHA method, and the time required to complete the test was only 4 h for the IAHA method, compared with 20 h for the CF method. The increased sensitivity of the IAHA test should permit its use for determination of immunity, as well as for serodiagnosis of recent infections.


Syncytia inhibition by immune lymphocytes: in vitro test for immunity to canine distemper.

A simple and rapid (24-h) assay for peripheral blood lymphocyte-associated immunity to canine distemper virus (CDV) is described. The test is based upon leukocyte-associated inhibition of CDV-induced syncytia formation in Vero cells. The technique quantitates the response morphologically, thereby eliminating the requirement for release of radiolabeled compounds. Positive results were determined from specific-pathogen-free and gnotobiotic dogs exposed to CDV via hyperimmunization, vaccination with a modified live virus vaccine, and after virulent virus infection. Preinoculation lymphocytes and lymphocytes from non-immune dogs did not inhibit CDV-induced syncytia formation. Maximum responses were observed 7 to 21 days after initial exposure and declined thereafter. The method can be used to further investigate the role of immune lymphocytes in the recovery from CDV infection.


Evaluation of a new microquantity blood collector.

A new device for collecting small amounts of blood from laboratory animals offer distinct advantages over standard syringes during the collection of serum or plasma.


The morphology of cirrhosis. Recommendations on definition, nomenclature, and classification by a working group sponsored by the World Health Organization.

This memorandum provides guidelines on the definition, nomenclature, and classification of cirrhosis, chronic hepatitis, and hepatic fibrosis. These are considered according to morphological characteristics and aetiology. It is hoped that this system will serve as a standard for diagnostic, research, and epidemiological purposes. The relationship of cirrhosis to liver cell carcinoma is briefly discussed and the possible morphological markers of an increased risk of malignancy are defined.


Hepatitis in clinical laboratories 1975-76.

The survey of British laboratories in 1970-74 was continued by a questionnaire to members of the Association of Clinical Pathologists covering the next two years. Only nine cases of hepatitis (three of hepatitis B) were reported. The annual attack rate of 34 per 100 000 was only one-quarter of that in earlier years. The findings suggest that control measures have had considerable success.


Autoclaving practice in microbiology laboratories: report of a survey. The Public Health Laboratory Service Subcommittee on laboratory autoclaves.

The performance of autoclaves in 27 laboratories, operated in accordance with the normal routine of local practice, has been monitored using thermometric equipment. Sterilising performance was unsatisfactory on 10 of 62 occasions, and cooling was inadequate on 52 of 60 occasions.


Use of polyvinylpyrrolidone in the testing of staphylococci for sensitivity to methicillin and cephradine.

The use of polyvinylpyrrolidone, an inert polymer resembling plasma proteins in its colligative effects, in the testing of micrococcaceae for sensitivity to methicillin and cephradine is described. Generally results are quite comparable with those of conventional methods. The absence of any inhibitory effect of the polymer compared to sodium chloride, and its physiological inertia compared to sucrose, along with its suitability for sterilisation by autoclaving are seen as advantages. It is suggested that the use of this substance may give results which are more applicable to the in vivo situation. This may apply particularly in the case of cephradine.


Effect of clindamycin and lincomycin therapy on faecal flora.

Bacterial counts were carried out on the faeces of 160 patients receiving clindamycin or lincomycin treatment for bacterial infections. In all the patients the total bacteroides count was significantly reduced while strains of Enterobacteriaciae, yeasts, and streptococci were correspondingly increased. Severe diarrhoea developed in 25 (16%) patients, but this could not be related to a change in faecal flora. Diarrhoea was most common when clindamycin was given prophylactically, women were more affected than men, and the incidence was highest in those aged over 60 years. No cases of pseudomembranous colitis were seen. Although clindamycin is a valuable antibiotic for treating established severe anaerobic bacterial infections it should be used cautiously in elderly patients.


Urine xanthine oxidase activity in urinary tract infection.

Xanthine oxidase (XO) activity was found to be negligible in sterile human urines (less than 480 units, as presently defined, per litre). Significant XO activity was found in all urines containing more than 10(5) bacteria/ml, except for urines infected with Staphylococcus aureus, in which XO activity ranged from 347 to 714 units per litre. Plasma XO is not transferred to the urine, as demonstrated by the negligible XO activity found in sterile urines from patients with raised plasma XO activity. Determination of urinary XO activity is a suitable procedure for the detection of urinary tract infection.


Urinary excretion of glycosaminoglycans in disseminated neoplasm.

Urinary glycosaminoglycan excretion was studied in 24 cases of disseminated neoplasm, 12 of which had unequivocal evidence of skeletal involvement. Urinary hydroxyproline, cetylpyridinium chloride (CPC)-precipitable uronic acid, and CPC-precipitable hexosamine were expressed as a ratio to urinary creatinine. Glycosaminoglycans contained in urine concentrated x 1000 by vacuum-dialysis were separated by electrophoresis on cellulose acetate and stained with alcian blue. Of the 12 cases with clear evidence of skeletal involvement, eight (66%) showed elevation of serum alkaline phosphatase, five (42%) showed elevation of urinary hydroxyproline, and three (25%) showed elevation of urinary uronic acid. It is concluded that urinary uronic acid is not a sensitive index of skeletal involvement in disseminated neoplasm. The most striking feature of the study was the identification of a well-defined fraction indist inguishable from hyaluronic acid in seven (58%) of the cases with evidence of skeletal involvement. Hyaluronic acid is not normally identifiable in adult human urine. The hyaluronic acid excretors showed more consistent biochemical evidence of bone disease (elevation of serum alkaline phosphatase and urinary hydroxyproline) than the non-excretors. The possibility that the urinary hyaluronic acid is derived from degradation of skeletal hyaluronic acid is discussed. An alternative explanation is that the hyaluronic acid is derived from neoplastic cells as part of a reversion of glycosaminoglycan synthesis to a more ;fetal' state, a glycosaminoglycan counterpart of the production of oncofetal antigens by neoplastic cells.


Cell receptor studies on six anaplastic tumours of the thyroid.

Cell suspensions from six anaplastic thyroid tumours were studied for expression of lymphocyte and macrophage surface markers, and results were correlated with electron microscopy, clinical extent of disease, and response to radiotherapy. The clinical presentation of the disease was similar in all six patients. In five cases, many of the cells showed surface immunoglobulin. Electron microscopy was available on three of these and showed appearances in keeping with malignant lymphoma. The single case whose cells did not show surface immunoglobulin had an entirely different ultrastructure and was probably a carcinoma. This case was the only one that did not achieve complete remission with radiotherapy and the patient died from extensive local recurrence. It is concluded that receptor techniques are of value in distinguishing between malignant lymphoma and other anaplastic tumours of the thyroid, particularly when the results are correlated with histology.


Echinococcus granulosus: specific quantification of the two most immunoreactive antigens in hydatid fluids.

Preparations of the two most immunoreactive Echinococcus granulosus antigens (antigens 4 and 5) from sheep hydatid fluid, purified by a simplified method, and monospecific antisera against antigens 4 and 5, prepared by a new procedure, were used to measure the antigenic concentrations of antigens 4 and 5 in swine, sheep, and human hydatid fluids from pulmonary or hepatic cysts. Two bovine samples and two commercial preparations were also tested. The concentration of both antigens was significantly higher in sheep and human hydatid fluids than in swine hydatid fluid. The antigenic content of the two bovine samples and of the two commercial preparations was below the sensitivity level of the method employed. Independently of the species tested, the amount of Echinococcus antigens was greater in hepatic than in pulmonary cysts. The ratio between the concentrations of antigens 4 and 5 was constant at about 1:10 in the samples from various organs and from different species. When there were enough samples for statistical analysis a linear correlation was found between the contents of these two antigenic components but there was none between the amounts of proteins and the antigenic concentrations in the single cysts. Sheep hydatid fluid must therefore be considered the best source of antigenic material for diagnostic purposes even though in human cysts the antigenic fraction is less contaminated by serum proteins. We describe a reliable method of standardising antigenic material for the immunodiagnosis of hydatid disease.


Comparison of radial haemolysis with haemagglutination inhibition in estimating rubella antibody.

Radial haemolysis (RH) for assaying rubella antibody was studied in parallel tests with haemagglutination-inhibition (HAI) on 1102 sera from adult women. Simple to perform and economical in reagents, the RH test is as sensitive as the HAI test and is a useful method of screening antenatal and other sera for rubella immunity. It detects IgG but not IgM antibody.


Coagulation and fibrinolytic activity of cerebrospinal fluid.

Fibrin/fibrinogen degradation products (fragments D and E) were detected in cerebrospinal fluid in 23.4% of 252 patients admitted to a neurological/neurosurgical unit. Other coagulation proteins of low molecular weight (plasminogen and factor IX) were also present but larger proteins (fibrinogen and factor V) were not. These findings are consistent with protein leakage across a blood-CSF barrier damaged by inflammatory, vascular, or neoplastic disease. Fibrin/fibrinogen degradation products in cerebrospinal fluid after subarachnoid haemorrhage may not, therefore, be a reliable index of increased fibrinolytic activity in the subarachnoid space and may be misleading when selecting patients for fibrinolytic blockade.


High mean red cell volume: its incidence and significance in routine haematology.

With the advent of electronic particle counters of the Coulter S type the mean cell volume (MCV) has become an integral and useful feature of the red cell profile. Abnormally high values, often of minor degree, are particularly common but their precise clinical significance may be difficult to establish. This study defines the normal range and determines the incidence and distribution of the high MCV in routine hospital practice. Two hundred consecutive adult patients with an MCV of 100 fl or more were identified from the Coulter S analysis of 6542 blood samples and the underlying cause was established in 80%. Some of the clinical and economic implications of these findings are presented and briefly discussed.


A quantitative analysis of the innervation of the pulp of the cat's canine tooth.

Although the cat's canine tooth has become the accepted model for the electrophysiological study of dental sensory mechanisms no examination of its innervation has been carried out at the electron-microscopical level. This study looked at the number and size distribution of both myelinated and non-myelinated fibers in the crown of the cat's canine. The material examined was prepared by routine methods and the measurements taken from electron-microscopical montages of pulpal cross-sections. The measurements were made using a Quantimet 720 image analysis system. In one complete cross-section 3,470 fibers were counted. Eighty-one percent of these were non-myelinated with a modal diameter of 0.35 micron. The modal diameter of the myelinated fibers was 2.5 micron. The relative preponderance of non-myelinated fibers increased from core to periphery. The largest myelinated fibers were concentrated in the core. The fiber size distribution was similar in the single complete and two partial sections examined. It is concluded that all the fibers in the crown of the cat's canine would be contained in the A-delta and C groups and that the strikingly large number of fibers present suggests that the peripheral pulp has a dense innervation, many of the cell processes found there being unsheathed axons.


Radioautographic investigation of gliogenesis in the corpus callosum of young rats. I. Sequential changes in oligodendrocytes.

The corpus callosum of young rats was examined to clarify the behavior of the three subtypes of oligodendrocytes (the large organelle-rich "light oligodendrocytes," the smaller and more densely stained cells referred to as "medium oligodendrocytes," and the even smaller and denser "dark oligodendrocytes"). It was hoped to find out whether cells of the three subtypes undergo division and how they are related to one another. 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to a first group of 19- to 20-day old rats weighing about 40 g, and to a second group of 25-day old rats weighing about 80 g. The animals were sacrificed at various time intervals from 2 hours to 35 days after 3H-thymidine administration. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. Two hours after 3H-thymidine injection, label is virtually absent from light, medium and dark oligodendrocytes, from microglia, and probably from astrocytes, but is present in about 10% of the immature glial cells, which include the poorly differentiated glioblasts and the partially differentiated oligodendroblasts and astroblasts. Hence, the cells undergoing DNA synthesis and mitosis in the corpus callosum are these three types of immature cells. During the week that follow the administration of 3H-thymidine, label appears in oligodendrocytes and astrocytes, which presumably have arisen from the initially labeled immature cells. The oligodendrocytes acquire label in a sequential manner: the light cells show label first and their labeling index reaches a peak at the seven-day interval; the medium oligodendrocytes become labeled next with a labeling peak toward the 14- and 21-day intervals and, finally, the dark oligodendrocytes with a peak around the 28-day interval. Analysis by the method of Zilversmit et al. ('42-'43) provides precise details on the sequence: immature cells presumed to be oligodendroblasts give rise to light oligodendrocytes which, after four to seven days, transform into medium oligodendrocytes which, after another 11 to 18 days, transform into dark oligodendrocytes. The dark cells may persist indefinitely or turn over at a very slow rate. It is concluded that oligodendrocytes arise from the last division of oligodendroblasts and develop in three main periods: a light stage lasting less than a week, a medium stage lasting about two weeks, and a very long lasting dark stage.


Radioautographic investigation of gliogenesis in the corpus callosum of young rats. II. Origin of microglial cells.

Microglial cells are absent from the corpus callosum of newborn rats. In the hope of finding out when and how microglial cells appear with age, 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to 5-day-old rats weighing about 15 g; and these animals were sacrificed at various time intervals from 2 hours to 35 days later. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. The corpus callosum of 5-day-old rats is composed of loosely arranged unmyelinated fibers and scattered cells. Among these cells, microglia are rare; there are a few astrocytes, many immature glial cells, rare pericytes, and 6--7% of phagocytic "ameboid cells" consisting of a few monocytes and many macrophages. In the animals sacrificed two hours after 3H-thymidine administration, label is present only in immature cells and "ameboid cells." As time elapses and the fibers of corpus callosum become myelinated, oligodendrocytes and, later, microglial cells appear. At the age of 12 days, microglial cells are present in substantial number; and by 19 days, the number doubles to reach a plateau. Many of the new microglial cells are labeled, e.g., 78.1% in 12-day-old animals (7 days after 3H-thymidine administration). The labeled microglial cells must have come from the transformation of cells that acquired label early, that is, from the immature cells or the "ameboid cells." The height of the peaks of labeling--59.8% at nine days for immature cells and 77.8% at 12 days for "ameboid cells"--points to the latter as precursors of the highly labeled microglial cells. Furthermore, the "ameboid cells" disappear as microglial cells appear and there are transitional elements between these two cell types. Cell counts suggest that about a third of the "ameboid cells" transform into microglial cells, while the others degenerate and die. Thus, the microglial cells which appear in the corpus callosum during the first three weeks of life result from transformation of the "ameboid cells"--a group of macrophages showing various stages of transition from monocytes. As for the occasional microglial cell appearing after the third week or in the adult, they presumably come directly from monocytes. In either case, monocytes would be the initial precursors.


Depth-marking the proximal negative response in the pigeon retina.

The depth distribution of proximal negativity in the pigeon retina was investigated with a dye-marking technique. Proximal negativity is recorded in three stages as the retina is penetrated. At and near the surface (level 1) the response is small and phasic: next, during "level 2" the one response exceeds the off response in amplitude: then, at "level 3" the off response exceeds the on response in amplitude. The dye marks were located in thin light microscope sections; they were about 20 micrometer in diameter, and were associated with larger areas of tissue damage. Level 3 dye marks were centred at the middle of the inner plexiform layer, and level 2 dye-marks were distributed more proximally. The amplitude maxima of the PNR are close to the middle of the inner plexiform layer, and are separated by approximately 15 micrometer.


Electron microscopic demonstration of a supraependymal cluster of neuronal cells and processes in the hamster third ventricle.

A supraependymal cluster of neuronal cells and processes consistently present on the floor of the hamster third ventricle was identified and characterized by means of correlative scanning (SEM) and transmission (TEM) electron microscopy. SEM revealed each cluster to be ovoid with the majority of its surface covered by dome-shaped protrusions and fine beaded fibers. A number of processes traveling individually or in groups also entered or exited from the cluster at its base. As these processes passed over the ventricular surface, they contributed to an extensive network on the floor and ventral aspect of the ventricular wall. Some processes terminated on the ependymal surface in bulbous endings while others penetrated the ependyma. The neuronal nature of these clusters and their associated processes was confirmed at the TEM level. The dome-shaped protrusions visible on the surface of the cluster in SEM corresponded to apical surfaces of neurons confined to the peripheral aspect of a core of loosely arranged processes. These cells exhibited a prominent nucleolus, stacks of rough endoplasmic reticulum (RER), polyribosomes, Golgi cisternae, mitochondria and microtubules (MT) and gave rise to dendritic processes which extended into the core. These dendrites gave off branches at acute angles and contained polyribosomes, single cisternae of RER and evenly spaced MT. Other profiles of processes within the core shared these characteristics, suggesting that they also were branches of the peripheral cells. Axons present within the core and on the cluster's surface exhibited vesicle-filled varicosities which frequently established synaptic contact with the peripheral cells and their processes. The presence of an intraventricular cluster of neurons which potentially communicates with centers extrinsic to the ventricle may have important implications in the hypothesized role of cerebrospinal fluid and tanycytic ependyma in the neuroendocrine regulation of anterior pituitary function.


Converging projections from the mediodorsal thalamic nucleus and mesencephalic dopaminergic neurons to the neocortex in three species.

Previous studies in the rat have shown that the neocortical dopaminergic afferents, originating in the mesencephalon, terminate in those areas of the frontal lobe which receive projections from the mediodorsal thalamic nucleus i.e., the prefrontal cortex. In order to clarify whether this overlap is accidental for the rat or a consistent feature of several species we have compared the projection areas of the ventral tegmental area and the mediodorsal thalamic nucleus in three species, rat, opossum and tree shrew, using HRP injections in combination with glyoxylic acid histofluorescence method. The results have shown, first, that the area innervated by the mediodorsal nucleus of the thalamus is localized in a different part of the frontal lobe in each species: dorsolateral in the opossum, anteromedial, polar and suprarhinal in the rat and frontopolar in the tree shrew. Secondly, this area alone in each species receives projections from the ventral tegmental area. Thirdly, this area alone receives a dense innervation in the deep cortical layers by fluorescent fibres probably containing dopamine. The neighbouring neocortical areas receive afferents neither from the mediodorsal nucleus of the thalamus nor from the ventral mesencephalic tegmentum; their catecholamine innervation is mainly confined to the superficial layers and appears to be of noradrenergic nature. Although the techniques used did not allow a precise determination of the borders of the two projection areas and, therefore, the exact degree of overlap, it appears that mesencephalic dopaminergic innervation is a characteristic feature of the prefrontal cortex in the mammalian brain.


Growth pattern of axons in the optic nerve of chick during myelogenesis.

The purpose of this experiment was to study the diameter of axons at the time of the initiation of myelin and the pattern of growth of axons in the optic nerve of the chick. Embryos between 15 and 20 days and chicks 3, 5, 22 and 60 days of age were studied on the electron microscopic level. Based on axon diameter a unimodal distribution of unmyelinated axons is present through day 20 of incubation with a mean of approximately 0.35 micrometer. This population is represented through 22 days of age but from day 3 on, a second distinct population of unmyelinated axons is present which has a mean diameter that is approximately twice that of the smaller unmyelinated axons. All axons do not increase simultaneously in diameter but once growth starts, the unmyelinated axons apparently double in diameter at a relatively rapid rate prior to myelination. On incubation day 17 less than 1% of the axons in the optic nerve is myelimated. The number of axons in this group and their diameter (mean approximately 1.2 micrometer) remain relatively constant through day 3 but from days 5 through 22, two distinct populations of myelinated axons are present. By day 60, three distinct distributions of myelinated axons are present with mean diameters of 0.51 micrometer, 1.76 micrometer, and 3.90 micrometer. These populations represent approximately 20%, 67%, and 13% respectively of the total fiber population. As age increases the diameter of some myelinated axons is as small as or smaller than the unmyelinated axons at an earlier period in development. This suggests that factors other than axon diameter might be involved in the start of myelination. It appears that the increase in axon diameter does not occur in a continuous manner but in a saltatory manner from one size to another.


Studies of visual function and its decay in mice with hereditary retinal degeneration.

Functional implications of mouse hereditary retinal degeneration have been studied at the level of the superior colliculus and visual cortex in the C57BL/6J-le rd strain. On autoradiography at a light-microscopic level, following eye injection with radioactive compounds, central visual structures appeared normal. A slight reduction in ipsilateral retinal projection was probably related to reduced retinal pigmentation associated with the light ear (le) mutation. In recordings from visual cortex and tectum in rd mice older than five months the cells discharged with highly rhythmic maintained activity. This ongoing activity depended on retinal input, since temporary asphyxia of the eye stopped it immediately. The frequency of the rhythm was influenced by the anesthesia. In these older mice no visual receptive fields could be mapped, but in a few tectal recordings it was possible to suppress the maintained activity by diffuse, very intense illumination. As in normal mice, no auditory or somatosensory responses were observed in the visual cortex or upper tectal layers. In recordings from tectum before the age of three weeks retinotopic topography and receptive fields were normal. By day 24 no receptive fields could be recorded from parts of the tectum representing the central 90--100 degrees of the visual field, whereas within a peripheral ring responses were still roughly normal under photopic conditions. Over the following four months these peripheral responses faded away slowly. Incremental thresholds, especially in the scotopic range, were elevated, rising slowly to unmeasurable values. Similarly during dark adaptation the thresholds fell to values several log units above those reached in normal mice; these values of dark adapted thresholds in rd mice rose with age. This is consistent with morphological changes known to occur in the retina as a consequence, of the rd mutation the rods degenerating before the cones.


The histopathology of diaper dermatitis. Historical review.

The precise pathogenesis of one of the most common cutaneous disorders, diaper dermatitis, has not yet been completely established, perhaps in part because relatively little histopathological knowledge has been obtained. This paper summarizes the available information in this field, mostly obtained by early investigators.


Tinea versicolor: a scanning electron microscopic view.

Tinea versicolor (TV) infections were studied in the scanning electron microscope utilizing several different sampling and preparatory techniques. Scalpel scrapings provided information only on the morphology of individual spores and hyphae, while biopsies and cyanoacrylate adhesive slides demonstrated the topography of the disease as well as providing information on how the infection spreads and recurs. Critical point drying was necessart to maintain the fine ultrastructure previously described. The deep follicular location of the spores seen in this study probably explains the tendency for TV to recur. The follicular packing was specific, since the eccrine pores seem to be exempt from the infection.


Bone marrow involvement in mycosis fungoides demonstrated by needle biopsy.

A bone marrow needle biopsy was performed in 19 patients with mycosis fungoides (MF). Two patients, one previously classified as stage 4 and the other with diffuse erythroderma had pathological bone marrow findings. Seventeen patients showed no evidence of bone marrow involvement, 10 of these were in the plaque stage. Bone marrow biopsy can be of value in assessing the stage of the disease.


Dobutamine in the treatment of depressed cardiac function: a study in patients with ischaemic heart disease during the early post-operative period.

Dobutamine was administered by intravenous infusion to 10 patients with ischaemic heart disease who had a low cardiac output syndrome following abdominal surgery. The dosage of dobutamine started from 2.5 mcg kg-1 min-1 and was increased stepwise to 5, 7.5, 10, 12.5 and 15 mcg kg-1 min-1. Cardiac index increased significantly from 2.05 +/- 0.32 to 3.03 +/- 0.61. min-1 min-2 with 15 mcg kg-1 min-1. Heart rate was unchanged with 7.5 mcg kg-1 min-1 but increased significantly from 97.7 +/- 18.5 to 126.1 +/- 21.5 beats. min-1 with 15 mcg kg-1. min-1. Stroke index increased significantly from 21 +/- 4.4 to 25.4 +/- 5.1 ml m-2. beat-1 with 7.5 mcg kg-1 min-1. Pulmonary wedge pressure fell significantly from 14 +/- 2.9 to 9.1 +/- 2.4 mmHg with 2.5 mcg kg-1 min-1. Mean arterial pressure showed no significant change. No side effects were observed in these patients. We conclude that in patients with depressed cardiac function dobutamine at low doses of 2.5 mcg kg-1 min-1 decreases afterload and filling pressures. At the average doses of 5 - 7.5 mcg kg-1 min-1 stroke index and cardiac index are increased. At higher doses of 10 - 15 mcg kg-1 min-1 heart rate and cardiac index increase while stroke index fails to increase further.


Drug utilization in a general intensive care unit.

Drug utilization in 200 general ICU patients was retrospectively studied. One hundred and two different drugs were prescribed with a mean of 7.0 +/- 4.6 drugs/patient. The potential for polypharmacy and drug interactions is discussed, as is the cost of such therapy.


Bacterial contamination of pulmonary artery catheters.

The catheter tips of 152 patients, who were haemodynamically supervised by pulmonary artery monitoring or continuous cardiac output determination, were bacteriologically examined. 106 cultures remained sterile, 21 cultures revealed a growth of non pathogenic organisms. Staphylococcus aureus was cultured 15 times, pseudomonas aeruginosa 4 times, Citrobacter as well as Escherichia coli twice, and Klebsiella and Enterobacter once each. There was no statistically significant connection between dwelling period and contamination. Also diabetes mellitus or corticoid medication in high dosage had no significant influence on contamination rate.


The application of potassium selective electrodes in the intensive care unit.

Recent advances in analytical electrochemistry have provided highly selective potassium ion exchangers and robust polymeric membranes that can be used to measure the potassium activity in plasma or whole blood. The relationship between the activity measurement and the more familiar concentration measurement is explored. The ion selective electrode (ISE) has certain practical advantages and these have been studied in relation to their use in an intensive care unit. The application of these membranes to the continuous measurement of potassium in the circulation is discussed.


The electrocardiographic and clinical diagnosis of myocardial contusion.

The clinical features and treatment of 34 patients with myocardial contusion is presented. The value of specific clinical features for diagnosis is discussed. Guidlines for therapy are presented.


Analysis of the click-evoked brainstem potentials in man unsing high-pass noise masking.

Brainstem electrical responses (BSER) to 60-dB-SL click in noise high passed at various cutoff frequencies separated b 1/2-octave steps were recorded in normal-hearing adult subjects. By applying a derived response technique, narrow-band contributions to the BSER from specific portions of the basilar membrane were revealed. Latencies and amplitudes of the various waves in the derived BSER were recorded. Results indicate that nearly the whole cochlear partition can contribute to the brainstem response. The shifts in latency of waves I, III, and V and amplitude changes of waves I and III as a function of CF appear to be fully comparable to those of the AP. In contrast, the amplitude behavior of wave V as a function of CF is different from waves I and III depending upon frequency range. The discrepency in the behavior of wave V with respect to the earlier waves suggests some sort of neural reorganization at the level where was V is generated. The fact that there are contributions to the brainstem response from apical portions of the cochlea opens the possibility for extending the brainstem technique in assessing the higher cochlear turn function.


Two-tone suppression in auditory nerve of the cat: rate-intensity and temporal analyses.

Responses to two-tone stimuli were recorded from auditory-nerve fibers in anesthetized cats. One tone, the suppressor, was set at a frequency above characteristic frequency and was fixed in intensity. A second tone was set at an excitatory frequency and was varied in intensity. The suppressor tone, when set at a sufficient level, always reduced the response to the excitatory tone by an amount equivalent to a fixed number of decibels, regardless of the excitatory tone's intensity. Estimates of suppression magnitude were derived from shifts in rate-intensity function obtained when the suppressor tone was present relative to the functions obtained for the excitatory tone alone. When suppressor-tone intensity was increased, suppression magnitude likewise increased. When the two tones were increasingly separated in frequency, either by varying the excitor or by varying the suppressor, suppression magnitude decreased monotonically. Suppression behaved in the same manner regardless of whether suppresor tone was excitatory or nonexcitatory. When frequency separation was small enough and when both tones were above the neuron's characteristic frequency, responses synchronized to low-order combination tones could be elicited. These responses usually possessed different rate-intensity characteristics and resulted in estimates of suppression magnitude which were spuriously low. When frequency separation is normalized with regard to position of traveling wave maxima within the cochlear duct, the magnitude of two-tone suppression for a given suppressor-tone intensity is seen to be frequency independent.


The effect of amplitude envelope on the pitch of sine wave tones.

Psychophysical experiments show that the pitch of a short sine wave tone depends upon the amplitude envelope of the tone. Subjects find that the pitch of an exponentially decaying tone (1dB/ms) is higher than the pitch of a (20-ms) rectangularly gated tone of equal frequency. The percentage difference in frequency required to produce equal pitches with the two envelopes depends upon frequency fo: 2.6% at fo = 412 Hz, 1.4% at fo = 825 Hz, 1% at fo = 1650 Hz, and 0.7% at fo = 3300 Hz. The pitch change is insensitive to the relative intensities of the two tones. The spectra of tones with the two different envelopes suggest no obvious explanation for the pitch change. However, the weighted time-varying spectra for tones with two different envelopes evolve differently with time. Alternatively the pitch change can be derived from a modified version of the auditory phase theory of Huggins.


Dependence of loudness growth on skirts of excitation patterns.

Over a range of 50 dB, the loudness of a 100-Hz tone was measured in the presence of a broadband noise with a low-frequency cutoff at 200 Hz. The noise was varied in intensity along along with the tone so that the signal-to-noise ratio remained constant at either 0 or--10 dB. Listeners judged the loudness of the tone by loudness matching, magnitude estimation, and magnitude production. The noise markedly decreased the tone's rate of loudness growth but not the range over which loudness grows. The overall decrease in steepness of the 100-Hz loudness function was greater than that previously reported at higher frequencies. It is hypothesized that the decrease was greater because the spread of excitation at 100 Hz was more effectively contained than at higher frequencies. Support for this hypothesis is given by measures of intensity discrimination at 100 Hz.


Binaural detection at high frequencies with time-delayed waveforms.

Recent research has demonstrated that the binaural system can utilize ongoing interaural time differences for lateralization at high frequencies as well as at low frequencies. The requirement is that the signal be complex so that the time difference appears as a delay in the envelope of the waveform at one ear. Reported here are several masking experiments that examine detection performance with time-delayed signals or maskers. In the first experiment, the signal was a 50-Hz band of noise centered at 4000 Hz that was time delayed by different amounts on different blocks of trials; the masker was similar band of noise, presented diotically. Large masking-level differences (MLDs) were obtained for some values of time delay, but the MLDs did not increase monotonically within time delay as they should were envelope time delay the basis for detection performance. Subsequent experiments in which the masker was time delayed and the signal was a diotic, high-frequency tone, revealed that detectability follows the autocorrelation function, and that MLDs as large as 24 dB can be obtained at 4000 Hz at time delays corresponding to negative values in the autocorrelation function. Examination of the signal-plus masker waveforms in these conditions reveals that ongoing interaural differences in level and cycle-by-cycle time exist in those conditions that yield MLDs. Since the time differences are small by usual standards, the basis for detection performance in these conditions appears to be the ongoing interaural level differences. In a final experiment, lateralization performance was measured for a time-delayed, complex waveform in the presence of maskers of various intensities. The results show that subjects are able to extract information about the time delay in the envelope even when the signal is added to a masker of equal intensity or greater. Thus, at the small signal-to-noise ratios used in our detection experiments, extraction of envelope time information was impossible, but also unnecessary, for detection was accomplished on the basis of another cue--most likely the ongoing interaural level differences.


Periodicity measures for repeated random auditory patterns.

Random auditory patterns were repliated according to defined rules, converted toauditory pulse trains, and presented to listeners. Three ined: the threshold fraction of repeated elements necessary for a fixed level of discrimination from a nonrepeated pattern; the accuracy of periodicity matching; and periodicity magnitude ratings. A short-term periodicity analysis over successive replications appears to be more representative of the behavioral measures than a long-term periodicity analysis. Of the signal measures of periodicity examined, the maximum autocorrelation, irrespective of delay, most closely reflects behavioral measures of periodicity strength.


Noise-induced threshold shift and cochlear pathology in the Mongolian gerbil.

Groups of six mongolian gerbils were exposed to two-octave (1414-5656 Hz) band noise for 1 h at 100, 110, and 120 dB SPL. Threshold shift at several frequencies was measured 0.5, 3, 6, and 12 h, and 1-28 days after exposure. Final thresholds were determined at least two months postexposure. Extensive threshold shift was observed in all groups 0.5 h after exposure (TS0.5h). Where threshold shift increased in the initial hours after exposure, such increases were correlated with eventual permanent threshold shift (PTS). Recovery of thresholds from 1-28 days after exposure was approximately exponential, and slowest at the edges of the exposure band. PTS was seen in the 110 and 120 dB SPL groups. With TS0.5h of 50 dB or less, no PTS resulted. With TS0.5h above 50-60 dB, eventual PTS increased linearly with a slope of about 1.25 PTS/TS0.5h. Cochlear damage was evaluated by light microscopy. The relationship between hair cell loss and PTS was consistent with an inner hair cell threshold about 40 dB higher than that of outer hair cells. It is suggested that recovery from noise-induced threshold shift may involve different mechanisms in the two types of hair cells.


Psychoacoustical aspects of synthesized vertical locale cues.

A new method of studying vertical localization is described whereby the high-frequency response of the external ear is synthesized by a computer program incorporating two variable latency delay and add processes. The resulting sounds were evaluated by presentation to subjects via headphones which bypass the external ear. Vertical movement of the sound source was perceived when the latency of one echo was changed dynamically between 100 and 300 microsecond and this effect required the presence of high-frequency signal components (about around 4 kHz). In further experiments, the variation of perceived elevation with echo delay was measured, and it is shown that the vertical locale of the apparent source is a monotonic function of echo latency in the 160-260 microsecond region. It is proposed that locale is decoded by a form of spectral pattern recognition, whereby the locale of the source is represented as a peak on an autocorrelation function. The time-axis values (between 160 and 260 microsecond) corresponding to these peaks are considered to correspond to vertical locale for elevations between roughly, 60 degrees above horizontal and 40 degree below.


Pitch and pitch discrimination of broadband signals with rippled power spectra.

A random-interval pulse train or wide-band noise when delayed (tau) and added back to itself (cos+) produces a stimulus with a consinusoidally varying (or trippled) power spectrum. The spacing between the peaks in the spectrum is equal to the reciprocal of the delay (1/tau). If the stimulus is delayed and added back at 180 degrees phase reversal (cos-), then a cosinusoidally varying power spectrum is generated whose spectral peaks are separated by 1/tau, but whose peaks are displaced by 1/2tau relative to the power spectrum of the cos+ stimulus generated with the same day, tau. These stimuli yield a pitch, such that the pitch of the cos+ stimulus is equal to approximately 1/tau and the pitches of the cos- stimuli are equal to approximately 0.9/tau and 1.1/tau. These pitch matching results were studied using a variety of matching stimuli and conditions. Following the identification of the pitches, a method of limits and a same-different procedure were used to study the pitch discriminability of both the cos+ and cos- stimuli. Delays (tau) ranging from 1 to 10 ms were studied covering a pitch range of 90-1100 Hz. The pitch discriminations associated with the cos+ and cos- stimuli were essentially the same for both the random-interval pulse train and the wide-band stimuli. These pitch-discrimination results are compared to those associated with a periodic pulse train. The research is also discussed in terms of discriminations of delayed sounds in reverberant environments. These are consistent with assumptions concerning the autocorrelation of the rippled stimuli within the dominant frequency region for pitch perception.


New cephalostat for speech physiology research.

A device for stabilizing a subject's head is described which does not depend on earposts, allows for voluntary evacuation by the subject, and can be constructed at low cost. This particular cephalostat is used currently for stabilizing the head in studies of speech production where lip and jaw transduction devices, mounted external to the subject, are employed.