A microfluorescent PAS method for the quantitative demonstration of cytoplasmic 1,2-glycols. Fluorescent microspectrophotometry using dichroic mirror vertical epi-illumination of tissue sections stained with the PAS reaction (periodic acid and pararosaniline Schiff reagent) provides a measure of the relative concentration of 1:2 glycols within and between tissue sections. In PAS reacted sections of agarose gel, pararosaniline Schiff fluorescence increases linearly as the concentration of agarose increases (r=0.97, p less than 0.05). The concentration of glycogen within liver as measured by a phenol-based tissue assay is linearly correlated with pararosaniline Schiff fluorescence of formalin fixed liver sections (r-0.87, p less than 0.05). These relationships are unaffected by alcian blue or hematoxylin. Heretofore the amount of color reaction as measured by densitometry at the pararosaniline absorption peak was claimed to be an unreliable indicator of the amount of reactive glycol present in tissue. Our observations indicate that when the concentration of Schiff reagent exceeds an empiric limit relative to available polysaccharides, the Schiff reagent-tissue complex reflects light at the excitation wavelength instead of fluorescing the emission spectra. This can be circumvented by using dilute pararosaniline-Schiff reagent, shorteining the staining period, and lowering the temperature of the staining medium. While routine PAS staining reactions are followed by washing in running water to develop the red color seen with broad spectrum illumination, water development is unnecessary for the dye-tissue complex to fluoresce. The fluorescent emission peak and the maximum excitation peak of both developed and undeveloped pararosaniline-Schiff-reagent-tissue complexes are 645--50 nm and 540--45 nm, respectively. These spectral characteristics are not changed by binding to oxidation products of different glycoproteins or polysaccharides. Intense exposure to room light, but not 100 repetitive short (0.13 s) exposures, causes partial photodecomposition. Quantitative assessment of cytofluorescence requires definition of the optical system used to measure emission. In the microspectrophotometer employed in this study, dichroic mirrors reflect light with variable efficiency depending on wavelength from the light source to the stage, and variably block light reflected or emitted from the specimen, serving as crude barrier filters. These dichroic mirror characteristics are influenced by the exact nature of the optical coating on the surface of each individual mirror. Since the optical coating on the surface of each individual mirror. Since the optical coating of similar mirrors may vary, the properties of individual mirrors must be considered in the interpretation of spectral data and in determining the proper optical conditions for quantification of cytofluorescence.


A new type of cytocentrifuge, the valve-centrifuge. A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves. The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation. Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei thorugh gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.


The use of X-ray microanalysis to demonstrate the uptake of the molluscicide copper sulphate by slug eggs. Copper has been localized in copper sulphate treated eggs of the slug Agriolimax reticulatus (Mü). This has been accomplished using a freeze-fracture technique, the freeze-dried halves of the fractured eggs being analysed in the scanning electron microscope, using both energy dispersive and wavelength dispersive X-ray microanalysis systems. The distribution of copper obtained using these methods has been compared with that achieved using a standard histochemical technique. Both techniques revealed that the copper is initially retained in the perivitelline membranes. The application of X-ray microanalysis to such studies is discussed.


Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes. A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both 3H-thymidine and 3H-urine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with 3H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoe scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains. On the other hand, with 3H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16--17 days' dry exposure the 3H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequency, has been found to have increased with the rapid method. No difference in grain density of labelling of 3H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.


[Laryngeal ruptures (author's transl)].

[Laryngeal ruptures (author's transl)]. Laryngeal ruptures are caused by vertical traction on the larynx and trachea, mainly by pushing the chin upon a dashboard or by so called whiplash trauma. Forteen cases could be classified in supraglottic ruptures, subglottic rupture and laryngotracheal ruptures. In supraglottic ruptures the epiglottis and parts of the ventricular folds are separated from the thyroid cartilage. In subglottic rupture the cricothyroid membrane is ruptured. The most common type is the partial avulsion of the trachea from the cricoid cartilage. In all types the laryngotracheal axis is kinked and scar tissue grows into the larynx. To avoid consecutive stenosis of the larynx in every case a straight dilator should be introduced as an internal splint.


[Sudden deafness as a consequence of rupture of a basilar artery aneurysm (author's transl)]. A sixty-eight year old female patient died from a subarachnoid hemorrhage three days after experiencing sudden hearing loss in her right ear. Autopsy showed that the cause of death was a large aneurism of the basilar artery. Temporal bone sections uncovered a massive infiltrate of erythrocytes about the nerves in the internal auditory canal. The blood cells had extended along the nerve bundles to enter Rosenthal's canal and the other fluid channels of the modiolus, and could be found within all perilymphatic spaces. The presence of hemosiderin within the inner ear correlates clinically with the onset of sudden deafness three days prior to demise, and is substantial evidence for the loss of hearing due to rupture of the basilar aneurism.


[Applied phoniatry. V. Speech defects due to abnormalities of jaw and dentition (author's transl)]. Dysgnathia (jaw and tooth anomaly, tooth misspacing, macillodento-facial anomaly) influences to a certain degree the development of defects of articulation especially sigmatism. Other factors, such as mild motor system inco-ordination and hearing defects, besides dysgnathia, are frequently observed. Speech therapy is necessary preceding as well as together with the maxillofacial treatment. The dental, prosthodontic and maxillofacial prostheses may likewise interfere with enunciation, but this will disappear spontaneously within 2 weeks or will be compensated for. If necessary the prosthesis will have to be altered.


[Acoustic and phoniatric investigations in esophageal speech (author's transl)]. The present study attempted to investigate the intelligibility of esophageal speech by utilizing speech audiometry. A tape recording of spoken numbers and monosyllabic words was made by laryngectomized patients who had learned esophageal speech. Normal hearing persons were then tested with this recorded speech for intelligibility. The threshold for detectibility of the spoken numbers in the study was found to be comparable for that of normal speech. However, the intelligibility curves of monosyllabic words was poorer when esophageal speech was employed. No improvement in  correctness of response in testees could be accomplished over a 65db intensity range. It is felt that this finding is based on the sound volume range of esophageal speech as modified by unfavorable signal/noise relationships.


[Nineteen years experience with surgical correction of disorders of swallowing and voice following vagus nerve paralysis (author's transl)]. An operation for the correction of swallowing and voice following paralysis of the vagus nerve is described. The operation is divided into four parts which are performed either in one or two stages. These include: 1. extramucosal myotomy of the pars fundiformis of the cricopharyngeus muscle; 2. correction of vocal cord paralysis by the rotation of the arytenoid cartilage and cartilage implant into the paralyzed vocal cord; 3. resection of the paralyzed wall of the pharynx; 4. fixation of the paralyzed soft palate to the posterior wall of the pharynx. Nineteen years experience with the four different parts of the operation are discussed.


Amphotericin B for the treatment of localized subcutaneous phycomycosis in the horse. Amphotericin B was used systemically or locally, or both, in the treatment of localized subcutaneous phycomycosis in horses. In 8 of 10 cases, the results were satisfactory. Intravenous treatment was well tolerated at an average starting daily dosage of 0.38 mg/kg, increasing up to as high as 1.47 mg/kg. It was concluded that the ideal treatment regimen would include early surgical removal of the lesion followed by daily intravenous and topical administration of amphotericin B, with periodic extirpation of small necrotic tracts as necessary.


Control of equine infectious anemia in horses in Hong Kong. Equine infectious anemia (EIA) has been enzootic in Hong Kong since the end of World War II. In 1972, a serologic survey of the horses at the Hong Kong Jockey Club indicated 23% prevalence of EIA. Disease control measures were instituted, and the spread of infection was reduced. In 1976, the prevalence of EIA was believed to be sufficiently low to implement procedures for eradication of EIA from all horses in Hong Kong. A correlation between EIA and poor performance of racehorses was demonstrated.


Failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. Failure in colostral immunoglobulin G (IgG) transfer was found in 9 of 87 Thoroughbred foals. Seven (78%) of these 9 foals acquired infections requiring therapy. Twelve of the foals had partial failure in colostral IgG transfer, and 3 of these had infections requiring therapy. The remaining 66 foals had normal transfer of colostral IgG, and only 2 had detectable infections. The failure of colostral IgG transfer was attributable to nursing problems in only one case. When presuckle postpartum colostrum was collected, 2 of 4 failures of colostral IgG transfer and 4 of 6 partial failures of colostral IgG transfer were explained by low colostral IgG content. Of 11 foals from various other sources and dying of infection before 2 weeks of age, 6 had failure of colostral IgG transfer (less than 200 mg IgG/100 ml serum), and 4 had partial failure (200-400 mg IgG/100 ml serum).


Cryotherapy of periocular squamous cell carcinoma in the horse. Squamous cell carcinoma around the eyes of 3 horses was treated with liquid nitrogen, using cryotherapy probes as the method of application. In 2 cases, there was complete regression of the tumor; in the 3rd case, remission and relief of discomfort were temporary.


Idiopathic monoclonal (IgA) gammopathy in a dog. Idiopathic monoclonal (IgA) gammopathy in a dog was differentiated from multiple myeloma, based on the lack of Bence Jones proteinuria, no osteolysis, and no plasma cell infiltration into bone marrow, as well as on the stability of the gammopathy for over 18 months. The dog was not treated but remained clinically normal for 18 months.


Survey of cat and dog ownership in Champaign County, Illinois, 1976. Age, sex, breeding, cost, source, and reproductive data on dog and cat populations were obtained for Champaign County, Illinois. Of 623 households from which completed interviews were obtained, 37.4% had 1 or more dogs, and 17.8% had 1 or more cats. The estimated population of dogs was 22,118 and of cats, 16,055, with a proportion to human population of 1 dog/7.4 persons and 1 cat/9.9 persons. The mean age of cats (2.98 yr) was about two-thirds of the mean age of dogs (4.64 yr); the median age of cats (1.8 yr) was about one-half the median age of dogs (3.7 yr). The most often mentioned origin of dogs was private pet owners; cats were most often reported as being offspring of currently owned cats. The proportion of households owning dogs and cats was higher in rural than in urban areas.


The striatins--new antibiotics from the basidiomycete Cyathus striatus (Huds. ex Pers.) Willd. Three crystalline antibiotics which we named striatins A, B, and C were isolated from the mycelium of the basidiomycete Cyathus striatus strain No. 12. The striatins are highly active against fungi imperfecti and a variety of Gram-positive bacteria, as well as against some Gram-negative bacteria. The molecular formulas as determined by mass spectrometry are C27H36O7 for striatin A, C27H36O8 for striatin B, and C25H34O7 for striatin C.


Evaluation of mycobacillin and versicolin as agricultural fungicides. III. Growth pattern and antibiotic production in soil by Aspergillus versicolor. Soil supports the growth of a jute pathogen Colletotrichum gloeosporioides but only to a limited extent that of its antagonist Aspergillus versicolor. The growth of the sensitive pathogen is considerably checked by the antagonist in mixed soil culture although versicolin production could not be demonstrated within the limits of assay. Both the sensitive and the antagonistic organisms grow well in soil-compost medium and versicolin production by the latter is also enhanced. The antagonistic effect of Aspergillus versicolor on Colletotrichum gloeosporioides is expectedly more marked in soil-compost medium than in soil medium.


Studies on the ionophorous antibiotics. VIII. Effects of monovalent and divalent cation ionophores on blood platelets. The effects of a monovalent cation ionophore etheromycin (CP-38295) and a divalent cation ionophore lysocellin on blood platelets function have been investigated. When platelets were incubated with 3 micronM of each ionophore, the simultaneous induction of secretion of stored serotonin and efflux of intracellular calcium occurred, but there was no detectable influx of extracellular calcium. With increasing concentrations, these compounds caused platelet aggregation accompanied by increased influx of external calcium. These results are consistent with the hypothesis that the liberation of calcium from the intracellular store mediated the platelet secretion reaction.


Antileishmanial activity of mycobacillin. Mycobacillin, a cyclic polypeptide antifungal antibiotic effectively inhibits the growth of promastigote form of the protozoal organism, Leishmania donovani, strain 81 in liquid medium. Oxygen uptake by intact cells of the protozoa with exogenous glucose is appreciably reduced within first 30 minutes in presence of the antibiotic at a concentration of 15 microng/ml. Appreciable leakage of intracellular 260 nm and 280 nm absorbing materials takes place from the protozoan cells incubated with the antibiotic at similar concentration.


A new alkaloid AM-2282 OF Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. AM-2282, a new alkaloid has been isolated from cultures of Streptomyces sp. AM-2282 by solvent extraction and silica gel chromatography. The compound exhibits a strong absorption maximum at 292 nm and shows antimicrobial activity against fungi and yeast. The LD50 of its hydrochloride (i.p. in mice) is 6.6 mg/kg. The molecular formula of AM-2282 has been determined as C28H26N4O3. The producing strain, AM-2282 was classified as a new species and the name, Streptomyces staurosporeus AWAYA, TAKAHASHI and OMURA, nov. sp. is proposed.


Studies on aculeacin. II. Isolation and characterization of aculeacins B, C, D, E, F and G. Six new antibiotics were isolated as the minor components related to aculeacin A from the culture broth of Aspergillus aculeatus M-4214 and named as aculeacins B, C, D, E, F and G. Their physico-chemical properties were analogous to those of aculeacin A and they showed significant activity against fungi. All of the minor components liberated palmitic acid on alkaline hydrolysis. Amino acid analysis showed that threonine and hydroxyproline are common constituents of aculeacins.


Further studies on ASN-136 and monoketo-organomycin cystaurimycin, a broad spectrum substance produced by partial enzymic digestion of monoketo-organomycin. The two antibiotics ASN-136 and monoketo-organomycin (MKOM) showed very close similarities in their UV, IR spectra and elemental analysis to those of tuberactinomycin and yazumycin respectively. Further chemical and enzymic studies revealed the novelty of the two former antibiotics. Partial enzymic hydrolysis of MKOM yielded a hydrolytic product of more potent inhibitory action compared with the parent antibiotic. Having cystine as the N-terminus and taurine as the C-terminus in its molecule, this enzymic degradation product was designated cystaurimycin. Performic acid oxidation of MKOM and of cystaurimycin improved their growth inhibitory effects on the test organisms used.


Effect of polyene macrolide antibiotics on invertebrate tissue culture cells. The effect of amphotericin B (AB) and amphotericin B methyl ester (AME) on viability and DNA synthesis in three insect cell lines, Trichoplusia ni (Tn) Carpocapsa pomonella 169 (Cp) and Aedes aegypti (Aa), has been evaluated. In all cases AME was less toxic than AB and inhibited DNA synthesis to a lesser degree than AB. However, the three cell lines differed in their response to the two polyene macrolide antibiotic preparations. Tn and Aa cell responded in a similar manner when exposed to either AME or AB, whereas Cp cells were more sensitive to both antibiotics than Tn or Aa cells.


Alveolar pressure and lung volume as determinants of net transvascular fluid filtration. We have investigated the influence of changes in alveolar pressure (PAlv) and in lung volume on the net transvascular fluid filtration rate (FFR). The preparation was isolated, perfused zone III rabbit lungs. In observation periods the outflow pressure was kept constant at a level generally causing net filtration. All pressures were measured relative to atmospheric. FFR was measured by continuous monitoring of preparation weight. Elevation of Palv at constant lung volume caused reversible reductions in FFR, also at constant capillary hydrostatic pressure (Pa-V less than 2 Torr). Increases in lung volume at constant PAlv caused reversible increases in FFR. When both PAlv and Ptp were increased a reduction in FFR was seen in the majority of cases. We conclude that at constant pulmonary arterial pressure, the size and the direction of the influence of positive airway pressure on FFR depend on the relative change in lung volume and in alveolar pressure per se. Under the present experimental conditions a rise in PAlv will be transmitted to interstitial fluid pressure and affect the transvascular fluid balance.


Effects of body temperature on ventilatory response to hypoxia and breathing pattern in man. The ventilatory response to hypoxia (PAO2 55 and 45 Torr) at each of four levels of PACO2 was studied in five healthy subjects before and after a rise in rectal temperature of 1.4 degrees C had been induced by means of a heated flying suit. At a given level of chemical drive both ventilation and mean inspiratory flow increased after heating, frequency relatively more than tidal volume. In isoventilation comparisons mean inspiratory flow was identical in normo- and hyperthermia, whereas the durations of inspiration (TI) and expiration (TE) were proportionately shortened. It is suggested that a rise in temperature shortens TI by affecting a central "clock" and that TE changes are secondary to changes in end-inspiratory volume. The euoxic CO2 response in hyperthermia was suggestive of multiplication between CO2 and temperature. Hypoxic sensitivity was significantly increased, indicating a temperature effect on the arterial chemoreceptors. The breathing pattern was in either temperature condition identical in euoxia and in hypoxia.


Airway responses to inhaled histamine in asymptomatic smokers and nonsmokers. Bronchia reactivity to inhaled histamine was assessed in asymptomatic cigarette smokers and in nonsmoking atopic and nonatopic subjects. The only prechallenge between-group difference was the ratio of maximal flow on 80% helium-20% oxygen (Vmax HeO2) to maximal flow on air (Vmax air) from partial expiratory flow volume curves at 25% vital capacity (25% VC PEFV): Mean +/- SEM for smokers 1.18 /+- 0.06, atopics 1.45 +/- 0.08, nonatopics 1.51 +/- 0.03. This suggests that prior to inhalation to total lung capacity, the predominant site of resistance at flow limitation was in smaller airways of the smokers and in larger airways of both groups of nonsmokers. Following inhalation of histamine, smokers and nonatopics had similar changes in lung volumes and Vmax air which were less than in atopics. The Vmax HeO2/Vmax air ratios at 25% VC PEFV increased in smokers and decreased in nonsmokers: smokers 1.48 +/- 0.08, atopics 1.22 +/- 0.10, nontopics 1.16 +/- 0.06. This suggests a predominant large airway response in smokers and a prominent small airway response in nonsmokers. These responses may reflect differences in the predominant site of aerosol deposition rather than in airway reactivity.


Forces applied to cranks of a bicycle ergometer during one- and two-leg cycling. An examination was made of the comparability of one- and two-leg exercise performed pedaling a stationary bicycle ergometer. The pattern of force exerted on both cranks was examined by means of a specially adapted ergometer which is described. The mean of the peak force in each cycle (MPF) was linearly related to work load (W) in both forms of exercise, and if account was taken of the doubled work output in two-leg cycling there was no significant difference between the MPF/W relationships; these are given by the equations one-leg: MPF (kg) = 11.23 + 0.065 (W in kpm/min) two-leg MPF (kg) = 10.76 + 0.032 (W in kpm/min). Calculation from the force records of the work performed on the cranks (WCR net) showed good agreement (r = 0.98, P less than 0.001) with the work load set on the ergometer. Analysis of the proportion of work done in leg extension and flexion phases of cycling revealed no differences between one- and two-leg exercise or between the right and left legs. The majority (approximately 80%) of Wer net being performed in leg extension is described by: Wer net (extension)) = 10.6 +/- 0.8 (W cr net total). In one-leg exercise (W greater than 900 kpm/min) the variation in rotation speed during a normal cycle ranged from +20 to --30% of the mean speed compared with +/- 10% in two-leg exercise.


Glucose-induced decrease in glucagon and pinephrine responses to exercise in man. Seven men ran at 60% of individual maximal oxygen uptake to exhaustion during beta-adrenergic blockade with propranolol or without drugs. After propranolol administration the increases during exercise in plasma glucagon and epinephrine concentrations as well as the decrease in plasma glucose concentrations were faster than in control experiments. When euglycemia was maintained by glucose infusion during beta-adrenergic blockade, glucagon and epinephrine responses to exercise, although not abolished, were markedly reduced. The diminution of the exercise-induced decline in glucose concentrations correlated significantly with the diminution of the glucagon as well as the epinephrine responses. Thus decreased glucose concentrations may significantly enhance the secretion of glucagon and epinephrine during prolonged exercise in man. Since the diminution of the glucagon response produced by glucose infusion was not accompanied by significant alterations in the levels of nonesterified fatty acid (NEFA) and glycerol, increased glucagon secretion does not seem to be a major determinant of lipolysis during exercise in man. During glucose infusion, glycogen utilization rates in muscle (n = 4) tended to decrease, whereas carbohydrate combustion rate and concentrations of norepinephrine, insulin, alanine, and lactate were unchanged.


Decrease of maximum work performance following ozone exposure. A bicycle ergometer was used to measure maximum exercise oxygen consumption following 2 h of intermittent exercise in an environmental chamber ventilated with air (FA test) or filtered air plus 0.75 ppm ozone (PO test). Thirteen adult males performed both tests according to a random sequence. The maximum attained VO2 declined 10% (P less than 0.01), maximum attained work load was reduced by 10% (P less than 0.01), maximum ventilation decreased 16% (P less than 0.01), and maximum heart rate dropped 6% (P less than 0.05) in the PO test. At the highest common work load, heart rate and oxygen consumption were similar and ventilation was slightly higher (P less than 0.05); however, frequency of respiration increased 45% (P less than 0.01) and tidal voluem fell by 29% (P less than 0.01) following ozone exposure. During maximum exercise, the respiratory frequency was similar in both tests, but tidal volume was 21% lower (P less than 0.01) in PO experiments. Decreases in vital capacity and FEV1.0 as well as cough and chest discomfort were also noted following ozone exposure. We conclude that the reduction of maximum attained VO2 is a consequence of ventilatory limitation of maximum effort, probably related to respiratory discomfort.


Panting in small mammals: a comparison of two marsupials and the laboratory rabbit. By use of a barometric technique, tidal volume (VT), minute volume (VE), respiratory frequency (f), and respiratory evaporative heat loss (Eex) were measured from conscious unrestrained potoroos (Potorous tridactylus), barred bandicoots (Perameles gunnii), and New Zealand white rabbits (Oryctolagus cuniculus) at temperatures in and above the thermoneutral zone (TNZ). Rectal temperature (Tre) and oxygen consumption were also measured. VT initially decreased with rising Ta, but in the potoroo and rabbit it then increased past the resting level. VE increased much more in the marsupials than in the rabbit, and higher Eex maxima were also found for the marsupials. The marsupials had high Q10's above the TNZ, and had a panting efficiency of 80%. The rabbits had a Q10 of 1.9 above the TNZ and 100% panting efficiency. The high VE and Eex and low panting efficiency of the marsupials may be due to their lower Tre which allows transfer of heat from the environment to the animal.


Oxygen storage in beavers.

Oxygen storage in beavers. Ten wild beavers were anesthetized with pentobarbital and total lung capacity (60.5 +/- 4.8 ml/kg), hemoglobin (12.4 +/- 1.5 g/100 ml), blood volume (6.5 +/- 0.8% body mass), and myoglobin (1.2 +/- 0.3 g/100 mg) were determined. These measured values were used to calculate total oxygen storage capacity. Even though the beaver is a good diver (15-min submersion time), its oxygen stores are not large and are about one-third the stores of a good marine diver, the harbor seal.


Frequency dependence of ventilation distribution in normal and obstructed lungs. Differences in regional pulmonary time constant may cause ventilation distribution to vary with breathing frequency and frequency-dependent changes in regional ventilation per unit volume might be expected. We measured the regional distribution of inhaled xenon-133 (133Xe) at 10 and 60 breaths/min in normal subjects and in patients with a clinical diagnosis of chornic bronchitis or asthma. Breathing frequency had no siqnificant effect on ventilation distribution in normals but in patients with chronic bronchitis ventilation decreased in the lower lung regions at 60 breaths/min compared with 10 breaths/min. In six asthmatic patients the lung regions demonstrating the greatest frequency-dependent reductions in ventilation, which were assumed to have the greatest time constants, also showed decreased ventilation-perfusion ratios (V/Q) measured by standard 133Xe techniques. Bronchodilator increased ventilation more than perfusion in these regions and thus V/Q ratio increased toward the normal range. These results imply that measurement of the frequency dependence of regional ventilation provides information about the relative distribution of airway obstruction, and that airway function in the most obstructed lung regions in asthmatics is improved following bronchodilator therapy.


Modification of renin and aldosterone response to heat by acclimatization in man. Plasma renin activity (PRA) and aldosterone concentration (PA) increased in eight men following a brief (30--40 min) heat exposure (50 degrees C dry bulb, 25 degrees C wet bulb) with light work. Sweat loss was less than 1% body weight. Plasma cortisol concentration was unchanged or decreased. In four subjects, a standard heat test was repeated in winter and summer (natural acclimatization). The increase in PRA and PA following heat exposure was less in summer than in winter. Four other subjects were artificially acclimated by daily work periods of 90 min at 50 degrees C for 7 days (artificial acclimation). Heat-induced elevation in PRA was considerably reduced by artificial acclimation, although postheat PA was reduced in only two of the four subjects. The small degree of sweat loss under the conditions of these experiments shows that circulating renin and aldosterone levels are increased in the heat even when a significant sodium deficit is not incurred.


Critical size of newborn homeotherms.

Critical size of newborn homeotherms. It is shown that for cylindrical and spherical bodies there is a critical radius below which the addition of any form of insulation to the body will increase rather than decrease the cooling of the body. It is proposed, therefore, that it would be thermally detrimental to newborn homeotherms to be born with a protective covering (fur or down) if their body size were less than this critical size, and consequently that the degree of natal covering is not necessarily related to the overall development of the species when the birth size is less than this critical size. A critical weight is derived from the critical radius for basically spherical animals which compares favorably with typical birth weights of various altricial homeotherms. The effect of the overall conductive-convective heat transfer caused by a basically cylindrical animal rolling up into a ball is also discussed.


Inversion of Fahraeus effect and effect of mainstream flow on capillary hematocrit. The well known Fahraeus effect (1929) states that if whole blood is allowed to flow from a large reservoir into a small circular cylindrical tube, the hematocrit in the tube is smaller than that in the reservoir, and the smaller the tube, the smaller will be the tube hematocrit. This is interpreted as a feature of particulate flow. We find that this relationship is not monotonic in a model experiment in which gelatin particles (circular disks) are suspended in a silicone fluid to simulate blood. When the diameter of the underformed cell is equal to or greater than the tube diameter, the volume fraction of the cells in the tubes increases to a value equal to or greater than that in the reservoir. Thus the Fahraeus effect has a point of inversion. Additional experiments show that the hematocrit in the tube could be greatly influenced by the flow condition outside the entrance of the tube. If the tube is perpendicular to the main direction of flow in the reservoir (as is the case of Barbee and Cokelet's experiment, or in most arteriole-capillary junctions), the velocity gradient and the velocity of flow in the reservoir just outside the entrance to the tube affects the hematocrit in the tube.


Effect of acute hypoxia on the hormonal response to exercise. The hormonal responses to submaximal exercise under normoxic and hypoxic conditions were studied in eight fit males, aged 22--28 yr, with mean maximal oxygen uptake of 4.4 +/- 0.7 l/min. Studies were performed in a hypobaric chamber, decompressed to a simulated altitude of 4,550 m (PIO2 = 83 Torr). The subjects exercised for 20 min at 750 kpm/min on a cycle ergometer. Venous blood samples were obtained at rest, during exercise and for 60 min after exercise. Plasma glucose, free fatty acids, lactate, cortisol, and serum growth hormone concentrations all increased more during hypoxic exercise than under normoxic conditions. Serum insulin concentration showed a small decrease under normoxic conditions, but decreased by 50% during hypoxic exercise, and was followed by marked rebound when exercise stopped. These changes suggest that energy substrate-hormone interrelationships are altered by hypoxic exercise, resulting in increased fat mobilization and increased gluconeogenesis.


Extension of pulmonary O2 tolerance in man at 2 ATA by intermittent O2 exposure. To study extension of O2 tolerance by interruption of hyperoxic exposure, as compared to previous studies of continuous oxygen exposure, five healthy volunteers were exposed to oxygen at 2 ATA on an intermittent schedule of 20 min breathing O2, alternating with 5 min on a normoxic N2-O2 mixture. The cycle was repeated until symptoms or signs of O2 toxicity caused cessation of the experiment. Tracheal irritation and burning on inspiration occurred after 6-9 "oxygen hours" of exposure and progressed to severe tracheobronchial burning sensation, chest pain, and dyspnea after 11-15 h of O2. Average duration of exposure was 13.7 O2 h, inducing a mean vital capacity decrease of 10.3%. The decrease began soon after onset of symptoms. With intermittent O2 administration, nearly a doubling of the average duration of actual oxygen breathing was required to induce marked vital capacity change (greater than 10%) as compared to the previous studies of continuous O2 exposure. The increased duration of tolerable O2 exposure in man resembles the extension of O2 tolerance known to occur in animals exposed to intermittent hyperoxia.


Role of the parasympathetic system in antigen-induced bronchospasm. Human subjects, sensitive to ragweed, inhaled aerosols of increasing concentrations of ragweed pollen extract in the presence and absence of pretreatment with inhaled atropine. The cumulative dose of antigen required for a 35% decrease in airways conductance was termed PD35. Atropine, 1.2 or 5 mg, increased airways conductance before antigenic challenge but did not alter signigicantly the PD35 for ragweed extract, although these doses of atropine inhibited airways responses to inhaled methacholine chloride and citric acid. We conclude that the cholinergic nervous system plays a modulating role in bronchomotor tone and responses to irritants, but that parasympathetic reflexes are not invariably a major component of human bronchial responses to inhaled allergen.


Comparison of measures of forced expiration. Theoretical relationships among a number of parameters were derived for idealized timed vital capacity (TVC) and maximal expiratory flow-volume (MEFV) curves to determine a minimal set of independent parameters. Normal pediatric subjects and those with cystic fibrosis were studied to verify these relationships experimentally. The average flow over the middle half (FEF25-75%) of the forced vital capacity (FVC) and flows at various exhaled percentages of the FVC (FEF50%, FEF75%), as well as moments of the TVC and MEFV curves were computed. From the TVC moments, a mean transit time (MTT) and an index of dispersion (ID) were also calculated. The minimum information needed to detect pulmonary mechanical changes associated with obstructive lung disease requires at least two reproducible measures: one related to the mean slope (e.g., FEF25-75%/FVC or MTT) and the other to the shape (e.g;, ID) of the effort-independent portion of the MEFV curve.


Human tecg changes during prolonged hyperbaric exposures breathing N2-O2 mixtures. In an effort to determine whether hyperbaric exposures while breathing N2-O2 mixtures have an effect on cardiac depolarization and repolarization, electrocardiograms of 10 divers participating in four N2-O2 saturation dives were analyzed. In all cases, a decline in heart rate was observed upon compression to saturation depth (20-30%); a slow adaptation and return of heart rate toward normal was observed in those dives where the depth and environmental parameters remained constant. twhenever excursion dives were performed, the heart rate responded by decreasing on deeper excursions and increasing on upward excursions. Hyperbaric bradycardia disappeared after 8 days at pressure during the saturation dives at 50 and 60 feet seawater gauge (fswg), but was still present at this time at 200 fswg. The magnitude of the hyperbaric bradycardia produced by excursion dives following saturation at depth was influenced by the state of adaptation of heart rate. Decompression was uniformly accompanied by a rapid increase in heart rate resulting in a significant elevation in the postdive period. Alterations in myocardial repolarization as evidenced by Q-T interval, ST, and T wave changes were observed. Development of slight right ventricular conduction delay compatible with right ventricular strain was noted in four of the divers during the two deepest dives to 100 and 198 fswg. During the latter dive, progressive decrease in P wave amplitudes and eventual loss of P waves resulting in an apparent nodal rhythm was observed in one diver. Multiple premature ventricular contractions occurred in another diver. These observations, along with the reports by other authors, suggest that the different variables associated with the hyperbaric environment--gas density, pressure, inert gas--have a definite effect on the pacemaker activity of the heart and myocardial depolarization and repolarization.


Extravascular lung water after extracellular fluid volume expansion in dogs. We have compared extravascular lung water after extracellular fluid volume expansion with that predicted from lung sucrose space measured in control dogs. In control lungs mean extravascular water:dry weight ratio was 3.81 +/- 0.16 (SD) (n = 5) and extravascular sucrose space/dry weight was 1.79 +/- 0.45 (n = 4). After acute expansion of extracellular fluid volume by 10% of body weight mean extravascular water:dry lung weight was 4.17 +/- 0.27 (m = 5), less than half the predicted increase to 4.63 +/- 0.19, suggesting some degree of protection. After 20% (n = 4), 30% (n = 2), and 40% (n = 1) expansion, no protection was demonstrated and there was considerable scatter of lung water at each infusion volume. When volume expansion increased pulmonary capillary intravascular forces (due to decreased protein osmotic pressure and increased hydrostatic pressure) by more than 20 cmH2O there was a linear increase in extravascular lung water with increasing intravascular forces. Three dogs did not conform to this relationship and had disproportionately large increases in lung water, possibly due to alveolar flooding.


Kidney structure and function of desert kangaroos. The structure and function of the kidneys of two species of desert kangaroos, the red kangaroo (Megaleia rufa) and the euro (Macropus robustus), were examined. Both kangaroos had glomerular filtration rates (GFR), renal plasma flows, and urine flow rates, when hydrated and dehydrated, which were lower than those of similarly sized eutherian mammals. The differences observed between the two species of marsupials were both structural and functional. The GFR of the red kangaroo was higher than that of the euro, under hydrated and dehydrated conditions, and this was correlated with the occurrence of larger and more numerous glomeruli, particularly juxtamedullary glomeruli, in the red kangaroo. Although the kidney of the euro had a greater relative medullary thickness than that of the red kangaroo, the latter had better urine-concentrating abilities. As opposed to this the euros reabsorbed significantly more urea from the filtrate when dehydrated (89.0%) than did the red kangaroos (69.2%). This ability of the euro to resorb more urea may be related to their tendency to overgraze their restricted home ranges during drought.


Extraction and radioimmunoassay of urinary aldosterone in the rat. Results from studies of the rat renin-angiotensin-aldosterone axis have been inconsistent and oftentimes conflicting among various laboratories and have led many investigators to regard the rat as a poor model with which to study this sytem. Many of these discrepancies have been shown to be due to the use of anesthetic agents in experimental protocols. Studies of unanesthesized animals indicate that the rat bears a remarkable resemblance to other mammalian species, but unfortunately the unanesthesized rat lends itself well only to acute studies, usually requiring killing the animal. Herein is described a method which permits the estimation of aldosterone excretory rates in intact, nonstressed, unanesthesized rats. The method is based upon the excretion and radioimmunoassay of urinary "acid-labile" or 3-oxo-conjugate of aldosterone. It is very sensitive, permitting the detection of less than 10 pg of aldosterone conjugate in extracted samples, and when compared to the double-isotope-dilution method, it is relatively inexpensive and much less tedious. Radioimmunoassays of rat urinary aldosterone excretion during 14.75 days of sodium depletion reflected a brisk increase in urinary excretion rate after day 2 and a concomitant reduction in sodium excretion that bears a remarkable resemblance to the excretory patterns described for man.


A modified forced oscillation technique for measurements of respiratory resistance. We present a modification of forced oscillation technique for automated determination of total respiratory resistance during inspiration. The modifications consist of a computerized signal averaging and an optimization technique in the assessment of the resistance value. Thereby a favorable signal-to-noise ratio is obtained, allowing very low superimposed pressure oscillations. The method is validated by comparison with a conventional esophageal balloon method, by estimating added mechanical resistances in healthy subjects and by measuring the effect of bronchodilation in asthmatic children. The coefficient of variation as obtained from day-to-day measurements was about 7%. Mechanical resistances, estimated as the difference in total resistance with and without external resistance, were within 7% of their values determined for the resistances alone. A significant decrease in resistance was obtained in each of the asthmatic children following bronchodilation.


Temperature control system for water-perfused suits. A system used to control skin temperature in human subjects wearing water-perfused garments is described, It supplies 8 l/min at 10 psi with water temperature controlled within +/-0.1 degree C. Temperature control is facilitated by a low circulating thermal mass and a fast responding heater based on a commercially available quartz heat lamp. The system is open so that hot or cold water can be added from the building mains to produce rates of change or water temperature exceeding 5 degrees C/min. These capabilities allow semiautomatic control of skin temperature within +/-1 degree C of desired wave forms. Potential hazards and associated safety devices are described.


Respiratory movements in the fetal guinea pig in utero. Intrauterine respiratory movements in the fetal guinea pig were detected from about 0.6 of term by the use of chronically implanted tracheal and amniotic catheters. Three distinct patterns were seen: 1) "slow" breathing recurring at a rate of 1-8/min; 2) "rapid irregular" breathing in which episodes last a few seconds to 40 min and the frequency of breathing is high (greater than 0.5 HZ) (the respiratory movements are associated with negative tracheal pressures of of 0.5-20 Torr); and 3) "rapid regular" breathing which recurs in bursts lasting 0.6-6.8 min with a maximum incidence of 3/h. Inspiratory efforts in the range of 10-20 Torr were uniform during a single episode. All three types of respiratory activity were continued into first stage labor, both during and between contractions, but were absent during second stage labor.


Hemodynamics of the lesser mouse deer, Tragulus javanicus. Mouse deer possess the smallest known erythrocytes. In contrast to the usual biconcave shape, mouse deer corpuscles are spherical which results in an appreciable increase in vitro blood viscosity. In the absence of compensation, increased viscosity should produce hemodynamic consequences. However, we find that cardiac output, arterial pressure, and total peripheral resistance are all within normal limits, while packed cell volume is considerably lower than normal. We suggest that reduced packed cell volume represents a compensation for the spherical shape of the corpuscles, serving to maintain blood viscosity and dependent hemodynamic parameters within normal limits. These results contrast markedly with the pattern observed in man where conditions which result in a high in vitro blood viscosity also have significant hemodynamic consequences. Mean values for three mouse deer were: body mass, 1.18 kg; packed cell volume, 29.8%; heart rate, 182 beats/min; cardiac output, 145 ml-(kg-min)-1., mean arterial pressure, 99 Torr; total peripheral resistance, 4.90 X 10(5) dyn-s-cm-5.


Parameters for assessing vibration-induced cardiovascular responses in awake dogs. The vibration parameters for assessing the response of the cardiovascular system to whole-body vibration were studied. Six awake, chronically instrumented canines were restrained with their spines vertical, and exposed to GZ sinusoidal vibration of 2-12 HZ for a constant peak acceleration amplitude of +-1.0 G. Vibration exposures of 30 s with intervening recovery periods of 2 min were employed. The following variables were measured: mean heart rate (MHR), stroke volume (SV), mean aortic flow (MAF), mean aortic pressure (MAP), the peak net force transmitted to the canine/body weight (PNF/BW), and the vibration platform frequency (ft), displacement, and acceleration. The percentage change from control (no vibration) of MAF varied linearly with PNF/BW for all cases. MAF also varied linearly with the log MHR/ft for the number of dogs which primarily changed MHR during the vibration exposures. The response of MAP was minimal in all cases, indicating a decrease in total peripheral resistance with increasing PHF.


Glucose loading and dehydration in the camel. Five female bedouin camels were subjected to large infusions of glucose, both when water was readily available and following 10 days of water deprivation. When the camels were hydrated the extra glucose was readily given off in the urine with only a slight increase in blood levels. Following dehydration, the blood glucose levels increased greatly while the urinary excretion was limited. Dehydration led to decreased blood insulin levels, while glucose infusion led to increased levels. The data show that the acclimatization of the camel to dehydration is not only a question of long-term adaption to desert conditions but that even following acute nonphysiological stress, i.e., glucosuria, excess loss of body water was prevented.


Changes in plasma glucose, FFA, corticosterone, and thyroxine in He-O2-induced hypothermia. Unanesthetized, male rats were exposed to normal air (NA), or NA and a 4 h-exposure of He-O2 (79% helium, 21% oxygen) at ambient temperature (Ta) of 22 or - 10 degrees C. Blood samples from each individual were taken from a chronically implanted carotid cannula at 1) preexposure, 2) during exposure, 3) 2.5 h after exposure, and 4) 19-20 h after exposure. Exposure to He-O2 at 22 degrees C caused an increase in plasma free fatty acids (FFA) and corticosterone of 45% and 49%, respectively, with little change in plasma glucose and thyroxine. Exposure to He-O2 at 10 degrees C for 3 h invariably induced hypothermia with body temperature (Tb) decreased to 23.7 +- 0.5 degrees C (N = 10). During hypothermia, plasma glucose, FFA, and corticosterone were significantly higher (P LESS THAN 0.05) than those at preexposure and those after exposure to NA at -10 degrees C. During spontaneous recovery from hypothermia, at Ta = 19 degrees C and NA, glucose, corticosterone, and thyroxine returned to normal, but FFA remained significantly higher than at preexposure. The ability of animals to rewarm spontaneously from hypothermia and the quick return of metabolic substrates and hormones to normal after rewarming indicates the preservation of regulatory mechanisms for metabolism at depressed Tb when hypothermia is induced by He-O2 and cold.


Influence of lung parenchyma on dynamic bronchial collapsibility of excised dog lungs. The main bronchi of excised dog lobes were obstructed 5-6 cm from their origin with beads, so that they did not communicate with the peripheral air spaces. With the lobe held at constant transpulmonary pressure, bronchial pressure-volume curves were recorded during both static and dynamic bronchial deflations. This was done both before and after dissecting the obstructed bronchus free of parenchymal attachments. The parenchymal contribution on dynamic deflation was much larger than the airway contribution: dynamic bronchial compliances in intact bronchi were 58 +- 8% and 65 +- 8% of the static at dynamic rates of bronchial deflation of 14 and 8 ml/s, respectively, whereas in dissected bronchi they were 72 +- 9% and 78 +- 9%. These relationships were not influenced by changes of transpulmonary pressure applied to the lobar parenchyma. The parenchymal effect on dynamic bronchial compliance was analyzed in terms of parenchymal interdependence and bronchial stress adaptation. We concluded that the presence of lung parenchymal attachments significantly reduced dynamic bronchial collapsibility as compared to static collapsibility.


Influence of body position on dynamic compliance in young subjects. We studied the effect of a postural change from the erect to the supine position on the unevenness of pulmonary ventilation in six normal subjects. Static pressure-volume curves had a similar shape in both positions but the supine curves were shifted such that all transpulmonary pressures were 5-8 cmH2O lower. Dynamic compliance (Cdyn) was measured in seated and supine postures at functional residual capacity (FRC) and also while the seated subjects maintained end-expiratory lung volume at the level of supine FRC. Frequency dependence of Cdyn increased relative to that measured in the seated position at FRC when the subject remained seated but decreased his lung volume, or when he assumed the supine posture. There was no significant difference between Cdyn measured in the supine posture and in the seated at supine FRC. We conclude that the increased frequency dependence seen in the supine posture is largely attributed to the associated decrease of lung volume rather than to position per se.


Effects of plasma ascorbic acid levels on heat acclimatization in man. Thirteen male volunteers were heat acclimatized for 4 h/day for 10 consecutive days. Three to four hours before each heat exposure, four of the subjects received an oral dose of 250 ascorbic acid, five received 500 mg ascorbic acid, and the remaining four a placebo. Rectal temperature, heart rate, and sweat rate were measured hourly during exposure. Venous blood samples were collected before each administration of drug or placebo. On days 1, 2, 3, 5, 8, and 10, blood samples were also collected just prior to heat exposure and after two and four hours of exposure. In the subjects receiving ascorbic acid, total circulating plasma ascorbic acid increased over the first three or four days to a plateau level some fourfold higher than in the subjects receiving the placebo. The plateau level was the same in the subjects receiving 250 mg and 500 mg ascorbic acid. The increased ascorbic acid concentration was shown to be associated with a reduction in total sweat output, independent of rectal temperature, and a reduction in rectal temperature, independent of total sweat output. The results indicate that ascorbic acid may be effective in reducing heat strain in unacclimatized individuals.


Effects of Valsalva and Mueller maneuvers on breath-holding time. Breath holding to the breaking point was studied at FRC in six healthy subjects in the sitting position. Breath-holding time increased with successive trials within experimental sessions in all subjects. To study the influence of Valsalva and Mueller maneuvers on breath-holding performance, sustained inspiratory or expiratory effort against an occluded mouthpiece was initiated 5 s before the anticipated breaking point, determined in previous trials. The subject tried to maintain a target mouth pressure of +20 or -20 cmH2O, displayed on an oscilloscope, for the remainder of the breath hold. Both types of maneuver consistently prolonged breath-holding time in all subjects. However, a control maneuver, in which the subjects squeezed rubber bulbs with their hands, was equally effective in prolonging breath-holding time. The results demonstrate the important influence of psychological factors on breath-holding performance and emphasize the need for caution in the interpretation of effects of "relieving maneuvers" on breath-holding time.


Effect of high-altitude exposure for 10 days on stroke volume and cardiac output. Resting stroke volume and cardiac output of 50 normal healthy sea-level residents (group A) were estimated by the noninvasive technique of electrical impedance plethysmography. They were then airlifted to an altitude of 3,658 m and serial estimations carried out at 0-4 h and 5-8 h and on the 2nd, 3rd, 4th, 5th, and 10th days. The subjects were brought back to sea level and studied for up to 5 days. Thirty permanent residents of high altitude (group B) and sixteen lowlanders temporarily resident at high altitude (group C) were also subjected to similar studies. It was found that resting stroke volume and cardiac output of group A started falling immediately on arrival at high altitude, reached the minimum on the 3rd day and tended to improved on the 4th and 5th day, but showed a secondary fall on the 10th day. The reduction in stroke volume in this group was not fully compensated by tachycardia. On return to sea level the cardiac output normalized immediately, the stroke volume on the 2nd day. At sea level goup A had values similar to group B and at high altitude to group C.


Urinary catecholamine excretion on induction to high altitude (3,658 m) by air and road. In a preliminary pilot study we had reported a significant difference in urinary catecholamine excretion between symptomatic and asymptomatic individuals inducted to high altitude by air. The present study covers slower induction by road; 25 lowlanders ascended from 1,800 to 3,658 m in 50 h and 33 similar subjects covered the journey in 6 h. They were studied according to the protocal used in the initial study. None of the 58 subjects inducted by road developed symptoms of high-altitude illness. Their urinary catecholamine excretion remained normal during the 10 days' stay at high altitude. These findings lend support to our earlier contention that there might be a relationship between increased sympathoadrenal activity and high-altitude illnesses.


Diet and starvation on the composition and calculated density of fat-free body mass. The density of the fat-free body mass (FFM) was calculated from carcass composition and density of body components in 148 growing rats under different dietary conditions. These conditions were controlled feeding with 20% and 5% casein diets during 6 and 9 wk, followed by 5 days of starvation, and after starvation with 2 wk refeeding with 20 and 5% casein diets. The results showed that the density of FFM was fairly constant. The means and standard deviations for all conditions were 1.0965 +/- 0.0057 and 1.0956 +/- 0.0075, for the 20% and 5% casein groups, respectively, with a grand mean of 1.0960 +/- 0.0066. It was concluded that the assumption of a constant density of FFM holds very well for many dietary and experimental conditions.


Effect of histamine on electrical and ion transport properties of tracheal epithelium. Previously we showed that the active transport of Cl-toward and Na+ away from the tracheal lumen creates an electrical potential difference (lumen negative) across the canine tracheal epithelium in vitro. The present study examined the effect of histamine on the electrical properties and ion transport of the canine tracheal epithelium in vitro. In six pairs of membranes, under short-circuit conditions, histamine (10(-4) M) significantly increased the net flux toward the lumen of Cl- from 1.7 +- 0.5 SE to 2.4 +- 0.6 microneq/cm2-h and Na+ from -0.4 +- 0.3 to 0.2 +- 0.3 microneq/cm2-h. The response to histamine was inhibited by diphenhydramine (10(-6) M), an H1-receptor antagonist, but not by burimamide (10(-4) M), an H2-receptor antagonist. These results are consistent with the hypothesis that an H1-type receptor mediates the increase of ion flux toward the lumen due to histamine. Increased ion flux toward the lumen may mediate water secretion into the airway lumen and have important effects on the physical properties of the liquid through which the respiratory cilia beat.


Correction of water content and solute concentration in blood during hemoconcentration. Eleven, healthy male students were subjected to an exhaustive bicyle ergometer exercise. Many parameters of hemoconcentration were examined in blood and urine samples which were collected before and after exercise. In an exercise lasting approximately 10 min, it was found that serum total protein (STP) would be a suitable parameter that could be used to calculate water content and actual decrease in water volume in blood due to exercise. Further, it is described how STP values before and during hemoconcentration can be used to eliminate the effect of plasma water loss from the determined values of various solute levels in blood.


Hemoconcentration during isotonic handgrip exercise. Three healthy males performed an intense isotonic handgrip ergometer exercise to exhaustion (approximately 10 min). Serum water, serum total protein (STP), and hematocrit were determined in the venous blood taken from the brachial veins in both exercised and nonexercised arms before exercise and 10 s, 1, 2, and 3 min after the termination of exercise. Although no sweat was observed, hemoconcentration occurred in both arms. The peak degree of water loss in the nonexercised arm, however, was delayed and lower than that in the exercised arm. Further, three healthy males were exposed to a sauna (76 degrees C) for 20 min to test the effect of sweating on hemoconcentration in the absence of physical movement. Venous blood was withdrawn from the forearm vein before the sauna, after 5 min in the sauna, and at 3 and 30 min during sitting recovery period. No hemoconcentration was observed during the course of the experiment.


Evaporation of sweat from sedentary man in humid environments. Physiological and sensory responses were observed in four male subjects while they were seated on a balance. The chamber temperature always equaled mean skin (Tsk)(34-36 degrees C), thus eliminating all sensible heat transfer. Ambient water vapor pressure (Pa) was increased in steps from 10 to 40 Torr. Tests at air velocities from 0.1 to 2.4 m/s were conducted with subjects nude and clothed. Esophageal temperature (Tes) and Tsk, heat rate (HR) and weight loss (M) were measured throughout the 2.5-h tests. After each 25-min humidity step subjects recorded their warm discomfort, sensation of sweating, and thermal sensation. Tsk, HR, and M were unaffected by humidity until critical Pa (P a crit) was reached above which M decreased and Tsk and HR increased rapidly. P a crit decreased with clothing and decreasing velocity. Tes remainded relatively constant throughout the tests. Sweating, discomfort and temperature sensations increased gradually with increasing Pa. Above P a crit sweating and discomfort sensations accelerated markedly. Mass transfer coefficients and clothing vapor conductance factors were evaluated. Skin wettedness at Pa crit decreased from 74% at 0.1 m/s to 35% at 2.4 m/s.


Axial forces in the bronchial tree.

Axial forces in the bronchial tree. Dog airways in situ are longer and extend more with inflation than when dissected out. The associated deformation of the parenchyma, from considerations of elasticity, appears to be small. The associated axial forces, to the extent they are transmitted along the tree, are governed by branching angles. Overall, average angles are such as would maintain constant axial stress along the tree and thus favor symmetrical lengthening. But locally, angles indicate considerable variation of transmitted stresses. Since radiography shows relatively homogeneous lengthening, we infer powerful local axial stabilization. In confirmation, dog airways were much less extended by locally applied forces in situ than excised. Do forced expirations apply substantial axial forces? In one subjects, during forced expiration, airway pressure fell abruptly near the carina but calculations showed relatively small axial forces applied to the airway. However, we speculate that large forces may be applied by airway plugs. We conclude that parenchyma stabilizes airway length against variably transmitted axial forces, and, perhaps, forces on airway plugs.


Infarct size and exercise capacity after myocardial infarction. Estimation of infarct size from serum creatine phosphokinase elevations of 22 acute myocardial infarction (AMI) patients was compared to their functional exercise capacity determined on a bicyle ergometer. Eleven of these patients entered a controlled exercise program for 3-4 mo and were subsequently retested. Aerobic power of the exercise group increased significantly (mean +- SE 1.42 +- 0.5 met) and was also significantly greater than an unpaired control group tested the same number of months after infarct. Patients grouped 2.5-4.5 mo post-AMI demonstrated a correlation between aerobic power and estimated infarct size (r = -0.68, n = 15). This correlation was higher (r = -0.84, n = 11) after 3-4 mo of a controlled exercise program.


Systematic design of cantilever beams for muscle research. Experimental studies of muscle contraction often involve difficult problems in the design of cantilever beams for movable levers, transducers, or mechanical supports. Equations are presented for the calculation of mass, inertia, stress distribution, strain, deflection curve, compliance, and resonant frequency of uniform or nonuniform cantilever beams made of structural materials of different density or elastic modulus. Formulas are listed for solid, thick-wall, and thin-wall uniform beams of rectangular and circular cross section. Physical properties including density, elastic and torsional moduli, stress and strain limits, thermal expansion coefficients, Poisson's ratio, and certain elastic-modulus-to-density ratios are tabulated for structural materials including common metals, glass, plastic, and wood. A graphical design procedure is presented based on a chart containing loci of constant beam parameter values as a function of beam length and height or diameter, for the simple geometries. The choice of structural material is discussed for design problems with typical constraints, and examples are given of the design of beams of nonuniform cross section. Methods for extending the design chart to other geometries and materials are included.


Atopic eczema: a clinical psychiatric study. Twenty subject (including patients with atopic eczema and normal matched controls) were subjected to extensive psychiatric interviewing and psychological testing. The atopic eczema patients were found to differ from the controls in early childhood experiences, in adaptation to aggressive conflicts, in themes of dreams and first memories as well as in conflicts and relationships to their extended families. A precipitant stress could be identified at the time of onset and exacerbation of the eczema. Significant psychiatric pathology was found in 60% of the atopic eczema patients. Psychological test results also indicated differences between the two groups with the MMPI  being the most discriminating test of those used.


A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum. A ferredoxin containing only one [Fe4S4] cluster was purified from Clostridium thermoaceticum. It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25. Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1). The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two [Fe4S4] clusters, and is similar to other ferredoxins with only one [Fe4S4] cluster. The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C. It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum. NADPH is used in the synthesis of acetate from CO2 in this organism.


Characterization of a spontaneously occurring mutant of the TOL20 plasmid in Pseudomonas putida MT20: possible regulatory implications. Pseudomonas putida MT20 carries a plasmid (TOL20) that codes for the enzymes responsible for the catabolism of toluene, m- and p-xylene to benzoate, and m- and p-toluate, respectively, followed by meta cleavage of the aromatic ring. Growth on 5 mM benzoate selects very strongly for (i) strains that have been cured of the plasmid and (ii) strains with an intermediate growth pattern (the B3 phenotype) that retain the ability to grow on toluene, m-xylene, and benzoate but are unable to grow on m-toluate. Both types of strains were selected because they are no longer able to oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or beta-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that one strain with the B3 phenotype, MT20-B3, has a regulatory mutation that prevents induction of the meta-pathway enzymes by benzoate and m-toluate, but which enables them to be induced by toluene and m-xylene, is presented. The plasmid in this strain, as in most of the others with the same phenotype, is nonconjugative. Analysis of MT20-B3, together with revertants of it and other noninducible mutants, has led to a model for the regulation of the plasmid-coded enzymes in MT20, in which it is proposed that the early enzymes for degradation of m-toluate and benzoate are positively controlled by two regulator molecules, one of which interacts with toluene and m-xylene as inducers and the other of which interacts with benzoate and m-toluate. It is argued that MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the second regulator molecule.


Distribution of xanthine oxidase and xanthine dehydrogenase specificity types among bacteria. A diverse collection of xanthine-metabolizing bacteria was examined for xanthine-, 1-methylxanthine-, and 3-methylxanthine-oxidizing activity. Both particulate and soluble fractions of extracts from aerobically grown gram-negative bacteria exhibited oxidation of all three substrates; however, when facultative gram-negative bacteria were grown anaerobically, low particulate and 3-methylxanthine activities were detected. Gram-positive and obligately anaerobic bacteria showed no particulate activity or 3-methylxanthine oxidation. Substrate specificity studies indicate two types of enzyme distributed among the bacteria along taxonomic lines, although other features indicate diversity of the enzyme within these two major groups. The soluble and particulate enzymes from Pseudomonas putida and the enzyme from Arthrobacter S-2 were examined as type examples with a series of purine and analogues differing in the number and position of oxygen groups. Each preparation was active with a variety of compounds, but the compounds and position attacked by each enzyme was different, both from the other enzymes examined and from previously investigated enzymes. The soluble enzyme from Pseudomonas was inhibited in a competitive manner by uric acid, whereas the Arthrobacter enzyme was not. This was correlated with the ability of Pseudomonas, but not Arthrobacter, to incorporate radioactivity from [2-14C]uric acid into cellular material.


Molecular sizes and relationships of TOL plasmids in Pseudomonas. Plasmid deoxyribonucleic acid was isolated from thirteen Pseudomonas strains judged on genetic criteria to carry plasmids coding for the degradation of toluene and m- and p-xylenes (TOL plasmids). Most strains carried a single species, but two strains carried two size classes, and cells of a third strain contained plasmids ranging in size from 25 X 10(6) to 202 X 10(6) daltons. Some plasmids could be transformed into a Pseudomonas putida strain to yield Tol+ progeny. Plasmids from 5 of the 13 strains were indistinguishable on the basis of size and gel pattern of fragments after endonuclease digestion.


Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldarius. Complex lipids from the thermoacidophilic facultative autotroph Sulfolobus acidocaldarius, as well as a strictly autotrophic isolate, were compared between cells grown on yeast extract and elemental sulfur. Lipids from both organisms grown autotrophically were nearly identical. Each contained about 15% neutral lipids, 35% glycolipids, and 50% acidic lipids. Glycolipids and acidic lipids contained C40H82-76-derived glycerol ether residues. Major glycolipids included the glycerol ether analogues of glucosyl galactosyl diglyceride (5%) and glucosyl polyol diglyceride (75%). Acidic lipids were comprised mainly of the glycerol ether analogues of phosphatidyl inositol (7%), inositolphosphoryl glucosyl polyol diglyceride (72%), and a partially characterized sulfate- and phosphate-containing derivative of glucosyl polyol diglyceride (13%). The lipids from cells grown heterotrophically were similar to those from autotrophically grown cells, except that the partially characterized acidic lipid was absent. In addition, the two glycolipids as well as the respective inositolphosphoryl derivatives were each present in nearly equal proportions.


Electron microscopic study of cell surface rings during cell division and morphogenesis of Arthrobacter crystallopoietes. The whole cell ultrastructure during cell division and morphogenesis of Arthrobacter crystallopoietes was monitored using electron microscopic techniques. Glucose-grown spherical cells were inoculated into succinate-based medium. In this medium, the organism undergoes a morphogenetic cycle consisting of elongation of spheres to rods, exponential growth as rods, and fragmentation of rods to spherical cells. Raised bands or rings that encircled the cells were evident on the cell surface of both sphere- and rod-shaped cells. Many rod-shaped cells possessed two or more rings arranged adjacent to each other in a parallel orientation. At each cell division a new ring was formed on both siblings. However, as predicted by the proposed model of unidirectional cell growth and by maintaining a ring from the previous generation, unequal numbers of rings were observed on sibling cells. Only one ring was visible on most of the spherical inoculum cells, but in some cases a second ring perpendicular to the other ring was observed. Parallel rings were found on spherical cells resulting from fragmentation or reductive cell division of rods during the stationary growth phase. Thus, these spheres could be distinguished from inoculum spheres containing a single ring or perpendicular orientation of rings. The number of rings per cell and arrangement of rings on the cell surface of sibling cells after cell division, but before cell separation, are discussed with respect to cell age, cell division, and sphere-rod-sphere morphogenesis of A. crystallopoietes.


Methanococcus vannielii: ultrastructure and sensitivity to detergents and antibiotics. Methanococcus vannielii is a strictly anaerobic motile coccus that possesses a tuft of flagellae. The cells are markedly sensitive to mechanical stress and are readily lysed by detergents, but the organism grows normally in media of low ionic strength. The absence of a typical cell wall, further suggested by resistance of M. vannielii to penicillin, cycloserine, and vancomycin, was confirmed by ultrastructural studies. Electron micrographs showed that the cell envelope lacks a peptidoglycan layer. On the outer surface there is a regular array of subunits similar to those of the glycoprotein envelopes of the halobacteria. However, the M. vannielii cell envelope, unlike those of the holobacteria, is unable to maintain a definite shape, and a high salt concentration is not required for its integrity.


Influence of oxygen on phospholipid production and colony formation in a nitrogen-fixing mutant of Azotobacter vinelandii. Colony dimorphism in a conditional nitrogen-fixing mutant of Azotobacter vinelandii was directly influenced by fixed nitrogen and oxygen partial pressure and may be related to the production of internal peripheral membrane.


Multiple forms of urease in cytoplasmic fractions of Ureaplasma urealyticum. The urease activity of Ureaplasma urealyticum was found to be located in the cytoplasmic fraction and consisted of multiple stable enzyme forms representing at least four biotypes.


Control of arylsulfatase in a serine auxotroph of Neurospora. A serine auxotroph of Neurospora requires exogenous serine to repress the arylsulfatase, suggesting that this enzyme is repressed by cysteine and not by methionine.


Initial characterization of hexose and hexitol phosphoenolpyruvate-dependent phosphotransferases of Staphylococcus aureus. The phosphoenolpyruvate sugar phosphotransferases of Staphylococcus aureus were surveyed biochemically to determine substrate range, inducibility and constitutivity, and requirements for soluble sugar-specific proteins. The substrate range is similar to that of the phosphotransferases of enteric bacteria, but the staphylococcal mannose and sorbitol systems are very inefficient. In addition, S. qureus has phosphotransferase activities for lactose and sucrose. The systems tested fell into two broad classes. Sugars for which there was substantial constitutive activity (fructose, mannose, sucrose, and glucose and its nonmetabolized analogues) did not require sugar-specific soluble factors for phosphorylation. Only in the case of fructose did growth in the presence of these constitutive sugars induce the corresponding phosphotransferase activity to higher levels. Kinetic experiments with each of these constitutive sugars yielded biphasic Hofstee plots; i.e., the kinetics were not characteristic of single enzymes. Preliminary experiments suggest that sucrose phosphorylation may involve the glucose and/or fructose systems. Truly inducible sugar phosphotransferase systems represent a second class; those for lactose and mannitol are the only members thus far identified. These systems are absent from uninduced cells, require soluble sugar-specific factors, and exhibit linear Hofstee plots. Sorbitol is apparently transported very poorly by intact cells but is an inducer of the mannitol system; it is phosphorylated efficiently in vitro by extracts of cells grown on either hexitol, but is taken up by intact cells at 0.1% of the mannitol rate.


Positional specificity of phospholipase B of Penicillium notatum. Phospholipase B[EC 3.1.1.5] of Penicillium notatum was shown to catalyze the following two reactions successively: diacylphospholipidlead to 1-monoacylphospholipid+fatty acid; and 1-monoacylphospholipid lead to glycerylphosphoryl-base+fatty acid. 1-Monoacylphospholipid was accumulated measurable in a reaction mixture containing methanol. An attempt to classify phospholipase B according to positional specificity is described.


Multiple components of endo-polyguluronide lyase of Pseudomonas sp. A lyophilized alginate lyase preparation obtained from dialyzed extract of sonicated Pseudomonas sp. cells was fractionated by gell filtration on a Sephadex G-150 column, and three alginate lyase [EC4.2.2.3] fractions, peaks I, II, and III, were obtained. They were remarkably thermolabile. The lyase fractions degraded two kinds of alginate fragments, a polygluuronide (SG), and a polyuronide consisting of both mannuronic and guluronic acid residues (SMG), as well as commerical alginate, but were virtually inactive toward polymannuronide fragment (SM). The modes of degradation of these substrates by the lyase fractions were endowise with different degrees of randomness. Attack by the peak I fraction was more random than those by peaks II and III. The main lysis products formed from SG and SMG by these layses were identified as mixtures of unsaturated tri- and monouronides. The unsaturated triuronide from SG was deltatugg and SMG yielded a mixture of deltaUGG and a poorly characterized unsaturated trimer, possibly deltaUMG. However, the patterns of monomer and trimer production by theselyase fractions changed in different ways during incubation.


Substrate specificity of endo-polyguluronide lyases from Pseudomonas sp. on the basis of their kinetic properties. Two endo-alginate lyases [EC 4.2.2.3] differing in their mode of degradation of substrates and practically free of polymannuronide lyase activity were partially purified from Pseudomonas sp. cells. Their substrate specificities were investigated for two different kinds of alginate fragments; a polyguluronide (SG) and a polyuronide consisting of mannuronic (M) and guluronic (G) acid residues (SMG). The effects of various salts and some organic compounds such as EDTA and p-chloromercuribenzoate on the degradation of the two substrates were similar. High concentrations of the substrates similarly inhibited the action ofthe lyases, giving a bell-shaped plot. A polymannuronide alginate fragment (SM) which was a substrate for polymannuronide lyase but was not attacked by these guluronide lyases also inhibited the degradation of SG and SMG. The overall degradation velocities of a mixture of SG and SMG by both lyases coincided with those calculated from the Michaelis-Menten formula. Based on the above results, it was concluded that SG and SMG are attacked by the same endo-polyguluronide lyase.


Fine structure of SMG alginate fragment in the light of its degradation by alginate lyases of Pseudomonas sp. An alginate fragment named SMG, consisting of mannuronic (M) and guluronic acid residues (G)(DP=25), was prepared from the partial acid hydrolysate of a commercial alginate. Two subfractions, SMG-ppt (DP=52) and SMG-sup (DP=18) were obtained from SMG by fractionation with MgC12 and CaC12. The M/G ratios of these alginate fragment were 1.4-1.9. Their lysis products by a pseudomonad alginate lyase [EC 4.2.2.3] preparation were fractionated by gel filtration, giving similar patterns. The major products in their digests were unsaturated monouronides (53-50%) and triuronides (30-35%). The former was identified as a delta4,5-hexuronic acid (deltaU) and the latter was identified as a mixture of delta4,5-hexuronosyl-(1 leads to 4)-beta-D-mannuronosyl-(1 leads to 4)-L-guluronic acid (deltaUMG) and delta4,5-hexuronosyl-(1 leads to 4)-alpha-L-guluronosyl-(1 leads to 4))L-guluronic acid (deltaUGG). The two unsaturated triuronides were present in roughly equal amounts. The presence of 4-O-alpha-L-guluronosyl-L-guluronic acid (GG) and 4-O-beta-D-mannuronosyl-L-guluronic acid (MG) or 4-O-beta-L-guluronosyl-D-mannuronic acid (GM) was also demonstrated inthe digest. Moreover, indirect evidence suggested nonreducing terminal deltaU residue and free deltaU in the digest to be derived more from M than G of the original SMG. Thus, it was concluded that more than one-third of uronic acid residues of SMG molecules may be composed of almost equal amounts of MG and GG sequences, most of which may be connected by M to form MMG and MGG sequences, respectively.


Studies on a microchemical method for determination of the degree of polymerization of neutral oligo- and polysaccharides. II. Application to oligosaccharides and glucans. A method devised for quantitative separation of trace amounts of alditols from their mixutes with large amounts of monosaccharides was applied in determination of the DPn of some oligosaccharides and glucans. The DP values obtained for oligosaccharides agreed very closely with those expected. The values obtained for polysaccharides were also consistent with those obtained by different chemical methods. This method is applicable to almost all neutral oligo-and polysaccharides with reducing end-groups, and can be use to determine the reducing end-groups in very small amounts of material.


Sites of action of the KCl-soluble protein in the stimulation of protein synthesis in sea urchin systems. The sites of action of the KCl-soluble protein (KSP) in the stimulation of amino acid incorporation were studied with cell-free systems from sea urchin embryos. The reduced form of KSP stimulated amino acid- and tRNA-dependent AMP exchange in the amino acid activation and initiation steps. Stimulation of the binding of phenylalanyl-tRNA (Phe-tRNA) to 40 S ribosomes by KSP was found; the reaction was considered to be the binding to the donor site of ribosomes, based on the results of puromycin treatment, so this stimulation is assumed to occur at the initiation step. All other steps of amino acid-dependent exchange of PPi and ATP as well as hydroxamate formation, amino acid acceptor activity of tRNA, the binding of aminoacyl-tRNA, mRNA, and synthetic polynucleotide to ribosomes, translocation and peptide bond formation, and the termination and release of peptides from ribosomes, were apparently insensitive to KSP. Cyclic variation was observed in the activities of amino acid- and tRNA-dependent AMP exchange with ATP and the binding of aminoacyl (acylated and nonacylated)t-RNA to ribosomes using 12,000 x g supernatant. The reduced form of KSP itself had AMP exhange activity in amino acid activation. The reaction involved was confirmed to be aminoacyl transfer from aminoacyl-AMP to tRNA. However, low activities were observed for the bindings of Phe-and acetylphenylalanyl (AcPhe)-tRNA to ribosomes. Small molecular SH-reagents reacted in a different way from reduced KSP both in the AMP exchange reaction and the binding of aminoacy1-tRNA to ribosomes.


Isolation of cytokinin binding protein from tobacco leaves by bioaffinity chromatography and its partial characterization. Cytokinin binding protein was isolated and purified from tobacco leaves by bioaffinity chromatography on a Sepharose column on which benzyladenine (BA), synthetic cytokinin, had been introduced as an affinity ligand by the cyanogen bromide method. The purified protein bound specifically to cytokinins; its binding was inhibited remarkably by the addition of BA and kinetin and slightly by adenine, but not by adenosine in vitro. The dissociation constant, Kd, of the protein-BA complex was about  4 x 10(-5)M. The profiles of SDS polyacrylamide gel electrophoresis and gel filtration indicate that the protein consisted of a single polypeptide chain. Amino acid analysis showed that the protein contained 4 basic, 6 acidic, and 25 neutral amino acids but lacked tryptophan. The molecular weight of the protein was determined to be about 4,000 to 5,000 daltons by gel filtration, SDS polyacrylamide gel electrophoresis and amino acid analysis. The coupling conditions of BA to Sepharose by the cyanogen bromide method are described and discussed.


The role of cathepsin B1 in the collagen breakdown of carrageenin granuloma in rats. The crude cathepsin B1 isolated from granulation tissue was separated  into two major peaks (P-2 and P-3 fractions) by chromatography on CM-cellulose. The changes in cathepsin B1 activity were studied at various phases of the development of granulation tissue. The cathepsin B1 activity of P-2 fraction, in contrast with that of P-3 fraction, increased with the passage of time after carrageenin injection and the cathepsin B1 activity of P-2 fraction was higher in the resorbing tissues than in non-resorbing tissues, suggesting that cathepsin B1 in P-2 fraction plays an important role in the resorption of granulation tissue, including collagen breakdown.


Amino acid sequence of the L-2 light chain of rabbit skeletal muscle myosin. The L-2 light chain (DTNB light chain) was separated from rabbit skeletal muscle myosin and the amino acid sequence determined. In order to study the primary structure of the L-2 lihe amino acid seqence determined. Then, to determine the arrangement of these tryptic peptides, enzymatic partial hydrolysis using 0.25 M acetic acid were carried out. The primary structure of the L-2 light chain thus obtained contains 168 amino acids.


Brain isoenzyme of creatine kinase. I. Purification of rabbit enzyme and production of specific antibodies. Creatine kinase (EC 2.7.3.2) of rabbit brain (BB) was purified to hemogeneity as judged by sedimentation velocity analysis in the ultracentrifuge. Nevertheless, the purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation also showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. At least some of the extraneous proteins appeared to arise from BB during storage. Subsequently BB was purified to hemogeneity by disc electrophoresis in polyacrylamide gel. It was excised from the gel and injected repeatedly with Freund's complete adjuvant into roosters. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. They did not, however, form immunoprecipitates with or inhibit rabbit muscle (MM) or rabbit hybrid (MB) creatine kinases. The antibodies were therefore specific for BB and did not react with MM or MB.


Brain isoenzyme of creatine kinase. II. Species specificity of enzyme and presence of inactive form in striated muscle of rabbit and man. Antibodies specific for the brain isoenzymes (BB) of creatine kinase were prepared by the injection of the highly purified rabbit enzyme into roosters. The antibodies were found to cross-react only partially with BB of rat, guinea pig, sheep, monkey, and man, demonstrating that these proteins are not entirely identical with respect to their antigenic sites. A greater degree of species specificity seems to exist for BB than for the muscle isoenzyme (MM) suggesting that BB may have undergone considerable evolutionary modification. Developmentally mature rabbit cardiac and skeletal muscles contained large quantites of an enzymatically inactive BB which can be detected immunochemically. It is antigenically identical with active rabbit BB but the two proteins can be separated by DEAE-cellulose chromatography. Enzymatically inactive BB is also present in human skeletal muscle. The presence of MM in muscle provides sufficient catalytic function and may have permitted the evolution of inactive BB in an additional (noncatalytic) metabolic role.


Brain isoenzyme of creatine kinase. III. Active and inactive forms in dystrophic muscle of vitamin E-dificient rabbit. Rabbits were fed a vitamin E-deficient diet for 7 weeks. Control rabbits, paired for weight, were pair-fed an identical diet supplemented with vitamin E. After 3 weeks the expermental animals showed a rise in serum creatin kinase activity which was attributable to the muscle isoenzyme (MM). No rise in creatine kinase activity or appearance of MM was noted in the serum of the control rabbits. Total creatine kinase activity and MM activity were reduced in the gastrocnemii of the experimental animals while the activities of the brain (BB) and hybird (MB) isoenzymes were increased. The specific activity of BB based on immunochemically determined BB protein was not different from normal in either the experimental or control group. Activation of pre-existing inactive BB is probably not the explanation for increased BB activity in the gastroncnemii of the experimental group.


Monosaccharide transport in protein-depleted vesicles from erythrocyte membranes. Treatment of human erythrocyte membranes with dilute alkali (pH 11.5) generates sealed, protein-depleted vesicles that can be isolated by density gradient centrifugation. The vesicles are 0.5 to 2.0 micrometers in diameter, and their membranes are predominantly oriented inside-out. The vesicles lack protein bands 1, 2, 5, and 6 (nomenclature of Steck, T.L. (1974) J. Cell Biol. 62, 1-19) of the erythrocyte membrane. L-Sorbose, a substrate of the monosaccharide transport system in erythrocytes, is transported by the vesicles. Based on comparisons between erythrocytes and vesicles with regard to specificity, temparture dependence, and effects of inhibitors, we conclude that sorbose uptake into the vesicles occurs by way of the monosaccharide transport system. The specific activity of the transport system in vesicles, as determined by initial rate measurements of sorbose uptake, averaged 58% of that in erythrocytes. This finding indicates that the major polypeptides of Bands 1, 2, 5, and 6 do not play an obligatory role in monosaccharide transport.


Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine. Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.


EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recogintion sequence. The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined. The replacement of cytidine in DNA with glucosylated hydroxymethylcytidine confers resistance to cleavage by the EcoRI endonuclease. Substitution of thymidine in DNA by hydroxy-methyluridine(a change in the methyl at position 5 of thymidine for a hydroxymethyl) lowers the maximal velocity of endonucleolytic cleavage 20-fold, but has no detectable effect upon the Km. Substitution of thymidine in DNA by uridine (a change in the methyl at position 5 of thymidine for a hydrogen atom) has no effect upon either the maximal velocity or the Km. The effect of these modifications upon EcoRI methylase activity was markedly different. DNA containing glucosylated hydroxymethylcytidine is methylated as well as normal DNA. DNA containing uridine or hydroxy-methyluridine, in place of thymidine, is much more poorly methylated than normal DNA. These different sensitivities of the EcoRI endonuclease and methylase to modifications in the pyrimidine rings of DNA suggest there are significant differences in the manner by which these enzymes recognize and bind to the canonical EcoRI sequence.


Complementation of peptides of barnase, extracellular ribonuclease of Bacillus amyloliquefaciens. Recovery of ribonuclease activity by complementation of peptides of barnase is reported. Activity is restored to barnase-(1-102), which lacks eight amino acids from its COOH terminus, by combination with peptides-(88-110), -(95-110), or -(99-108), and also with succinylated peptide-(88-110). The dissociation constants are about 8 X 10(-6) M for the first two combinations and little, if any, greater for the other two. Based on barnase-(1-102) concentration, up to 80% of native activity is obtained with peptide-(88-110) but only 5 to 20% with the others. The octapeptide-(103-110), equivalent to the residues missing from barnase-(1-102), does not complement barnase-(1-102), suggesting that an intact sequence about His-102 and Tyr-103 is required for activity. Barstar, the natural inhibitor of barnase, completely inhibits all activity of the complementing peptides.


Mechanism of aflatoxin B1 inhibition of rat hepatic nuclear RNA synthesis. This paper reports the evidence for the existence of multiple sites of action of aflatoxin B1 in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hours after aflatoxin B1 injection (0.3 mg/100 g body weight), rat hepatic nuclear and nucleolar RNA synthesis, in vitro, were inhibited 70 and 90% respectively. When total nuclear free and engaged RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 micrograms/ml), only alpha-amanitin-sensitive activity was reduced (50 to 70%) by aflatoxin B1. DEAE-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since aflatoxin B1 dramatically inhibited nucleolar RNA synthesis, but had little effect on RNA polymerase I activity per se, it is concluded, therefore, that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, aflatoxin B1 must also cause impairment of the nucleolar DNA template function.


Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum. Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.