Primary laboratory medicine.

A list of laboratory tests which should be available in a primary care facility is presented. The primary physician should be able to perform or obtain reports of blood smears, urinalysis, hematocrit, white blood counts, sedimentation and prothrombin information during the patient's visit. In general, those tests that are not practical to perform include those that are seldom used, that require extended time for performance, that are complicated and require special technical skills and that demand expensive and elaborate equipment and unstable expensive reagents. Equipment recommended for a primary laboratory is also listed. Tests to be run on each individual patient should be selected carefully because of the cost factor as there are limited financial resources for health care delivery.


Referrals and demand for specialist care in the Netherlands.

Three independent sets of data are analyzed to investigate the determinants of referrals from general practitioners to specialists, the demand for specialist care, and the rapid increase in referrals and use of specialists in the Netherlands. Individual determinants of demand are examined with data from a three-man group practice; macroanalyses are conducted on cross-section data from all service areas of the Dutch Sickness Fund Organization for 1971 and 1973 and cross-section time-series data from all provinces for the period from 1960 to 1972. Regression coefficients are estimated for the number of referrals to specialists and the amount of care given by specialists, with respect to explanatory variables that induce both socioeconomic and care-supply factors. Results are discussed in light of M. Feldstein's classification of three types of care, in which for some types the physician is seen as generating the demand for care. The authors conclude that demand for specialist outpatient care is in large part supply-determined and that general practitioners substitute for specialists; they also suggest that the dual physician-payment system in the Netherlands (capitation for legally insured patients and fee-for-service for privately insured patients) contributes to increased use of specialist care.


Test sequences in screening for breast cancer.

Published data of Stark and Way on the screening of 2,684 women at high risk of breast cancer are analyzed to arrive at a preferred sequence of screening tests. In the practical situation where palpation first signals a problem, the analysis suggests thermography to follow. Women with positive thermograms should then be biopsied, and those with negative thermograms should be mammographed. A positive mammogram calls for biopsy, and a negative one calls for close follow-up. For high-risk women whose breasts appear normal on palpation, a subsequent negative thermogram is not definitive enough to terminate investigation, but a negative mammogram after a negative palpation is enough evidence to waive further investigation for some time. A positive mammogram calls for immediate biopsy in any circumstance.


Nurse clinician productivity using a relative value scale.

The use of relative value units to measure the productivity of nurse clinicians in four primary care practices is described. Relative value points and equivalent dollar values assigned to services provided by professionals yielded a different assessment of productivity than that provided by a count of patient visits. The physician-nurse clinician teams studied were only 6 percent more productive than the physician-nurse teams when productivity was measured by the number of patient visits processed during an 8-hour period but were 26 percent more productive in terms of the value of services they produced per day.


Consultation frequencies in general practice.

Some models of the process by which individuals seek medical care suggest the negative binomial as the underlying distribution of the frequencies of consultations in a given practice. Data from the 1970-71 National Morbidity Survey of General Practice are used to test these competing models. It is shown that the negative binomial distribution successfully fits consultation frequencies in aggregate and in subdivisions according to age, sex, and duration of registration. In this article is is assumed that the consultation process has two components: the patient's decision to visit his doctor for a new illness and the follow-up visits that results from this new problem. Supplementing previous evidence that the distribution of episodes of new illnesses follows a negative binomial distribution, this article shows that consultation frequencies among individuals presenting with one new illness also follow a negative binomial distribution. A unifying model is required to synthesize these findings.


Location and cost of ambulances serving a rural area.

A location model is used to determine the most efficient (i.e., least-cost) number and location of ambulance facilities in a rural area. The model incorporates response time and service time standards into the analysis and indicates the trade-off between costs and various time standards. The financial feasibility of individual facility locations is then analyzed. The results indicate why many rural areas depend on volunteer or part-time purveyors of emergency medical transportation.


Self-sufficiency in a regional hospital system.

This article considers measures that indicate the degree of regionalization within a hospital system. In particular, regional independence is characterized with a "self-sufficiency" index. The basis of these measures is the use of facilities in one region by residents of another region, that is, interregional flows. Examples of the use of the suggested measure in the Nova Scotia hospital system are presented.


Three statistical models for estimating length of stay.

The probability density functions implied by three methods of collecting data on the length of stay in an institution are derived. The expected values associated with these density functions are used to calculate unbiased estimates of the expected length of stay. Two of the methods require an assumption about the form of the underlying distribution of length of stay; the third method does not. The three methods are illustrated with hypothetical data exhibiting the Poisson distribution, and the third (distribution-independent) method is used to estimate the length of stay in a skilled nursing facility and in an intermediate care facility for patients enrolled in California's MediCal program.


Effect of 2, 3-diphosphoglycerate on the solubility of deoxy-sickle hemoglobin.

We have examined the effect of 2, 3-diphosphoglycerate (DPG) on the solubility of deoxy-sickle hemoglobin (deoxy-Hb S) under conditions such that concentration, pH, and osmolarity of deoxy-Hb S solutions approached physiological. The range of DPG/Hb molar ratios encompassed the extremes found for this ratio in erythrocytes from individuals with sickle cell anemia. After monomer-polyer equilibrium had been established, the phases were separated by centrifugation and assayed for concentrations of Hb and DPG. DPG had no effect on the solubility of deoxy-Hb S. Furthermore, at DPG/Hb molar ratios less than one, there was no preferential incorporation of deoxy-Hb S containing bound DPG into polymers. At DPG/Hb molar ratios greater than one, concentrations of free DPG in monomer and polymer phases were virtually identical. Thus, under the specified equilibrium conditions, DPG is not a determining factor in the polymerization of deoxy-Hb S.


The molecular defects of alpha-thalassemia in the Filipino.

Molecular hybridization with synthetic radioactive DNA (cDNA) complementry to alpha globin mRNA sequences shows that, as in most other Southeast Asian populations, the alpha globin structural genes are deleted in Filipinos affected by the alpha-thalassemia syndromes. Thus, all 4 alpha-globin structural genes are deleted in homozygous alpha-thalassemia with hydrops fetalis, 3 and 2 structural genes are deleted in hemoglobin H disease and alpha-thalassemia-1 respectively.


An improved chromatographic procedure for quantitation of human fetal hemoglobin.

The DEAE-cellulose chromatographic procedure which uses glycine-KCN-NaCl solutions as Developers and a NaCl gradient to elute the hemoglobin zones was found to be useful for the quantitation of Hb F in samples containing Hb A, provided the level of Hb F exceeds 2 percent. The method has been evaluted through a study of artificial mixtures and of blood samples from patients with various disorders. The data have been compared with results obtained by four other procedures that are used for the quantitation of Hb F.


The linkage relationships of the beta and delta hemoglobin genes.

Three new families providing information on the linkage relationships between the delta and beta hemoglobin loci are reported. One of the families contains the first reported individuals having Hb S and Hb B2 in coupling. Analysis of the information found in the literture is compatible with a recombination frequency of 3 percent between beta thalassemia and the delta structural locus. This highly improbable frequency might be explained by either diagnostic errors or paternity problems in the pedigrees containing recombinants or by the conceivable possibility that certain thalassemia genes increase the probability for recombination in the chromosomal region containing the non alpha globin genes.


Absolute identification of Hb D-Los Angeles (beta121 Glu replaced by Gln).

This paper describes a method which permits the absolute identification of Hb D-Los Angeles without the need for N-terminal or amino acid analysis on peptide beta DT-13. The procedure involves the formation of the 5-oxo-2-pyrrolidinecarboxyl derivative of the N-terminal glutamine residue ot betaDT-13. This method, together with the use of cadmium acetate/ninhydrin staining, allows the identification of Hb D-Los Angeles, and other possible mutations of beta121, by peptide mapping.


Hemoglobin S-O Arab-alpha-thalassemia: globin biosynthesis and clinical picture.

A 22 year old American negro with mild anemia was found to be triply heterozygous for hemoglobin S, hemoglobin O Arab and alpha thalassemia. Hemoglobin A was not detected in the subject's hemolysate. The alpha thalassemia gene was expressed by an alpha/non-alpha synthesis ratio of 0.71 plus or minus 0.07 and was equally rates unbalanced in the peripheral blood and the bone marrow. The total radioactivity of the abnormal chains was equal, indicating equal rates of transcription and translation of the abnormal structural genes. Since the rate of synthesis of betaS and betaO was equal to the proportions of these abnormal chains in the peripheral blood, the abnormal gene products were equally stable. The mildness of the anemia, compared to that of the other reported S-O Arab double heterozygotes, may be due to the moderating influene of the alpha thal gene.


Biosynthetic and structural studies of hemoglobin in a patient with congenital dyserythropoietic anemia type I.

A 25 year old woman with congenital dyserythropoietic anemia (CDA) Type I is described. Typical morphologic abnormalities of the erythroid precursors in the bone marrow by light and electron microscopy, marked ineffective erythropoiesis and iron loading were present, Globin chain synthetic ratios as well as functional and structural studies on the patients hemoglobin were normal, ruling out the presence of thalassemia or a mutant hemoglobin which can both give rise to morphologic and clinical features similar to CDA. The laboratory findings on this patient and family members and a brief review of the literature are presented.


Binding of Protoporphyrin to hemoglobin in red blood cells of patients with erythropoietic protoporphyria.

Virtually all protoporphyrin in erythrocytes of patients with erythropoietic protoporphyria is bound to hemoglobin. The maximum of the fluorescence excitation spectrum of this protoporphyrin-hemoglobin complex shifted, with increasing concentration, from 405 nm to 389 nm. A similar shift was observed when titrating a solution of free protoporphyrin with hemoglobin. The Soret maximum of free protoporphyrin itself, on the other hand, was not concentration-dependent. These observations indicate that spectrofluorometric measurements do not allow conclusions concerning the mode of protoporphyrin binding to hemoglobin. Experiments on protoporphyrin exchange between the hemoglobins A, F and S reinforced the previously drawn conclusion that protoporphyrin is bound to hemoglobin at the heme-binding sites.


Adrenergic influences on the permeability of rabbit submandibular salivary glands to blood-borne horseradish peroxidase.

Horseradish peroxidase (HRP) administered close-arterially, has been found to enter rabbit submandibular saliva elicited by parasympathetic nerve stimulation. Adrenalin, superimposed on parasympathetic nerve stimulation, increased the passage of HRP into the saliva. Use of alpha- and  beta-adrenoceptor agonists, either separately or together, and use of alpha- or beta-adrenoceptor antagonists together with adrenalin indicate that both alpha- and beta-receptor stimulation is necessary for this increase in glandular permeability to occur. Histochemical assessment showed  that HRP had permeated the interstitial spaces of the gland and entered the spaces between adjacent parenchymal cells. However, in unstimulated glands it had only reached the lumina of striated ducts, but after adrenalin administration, peroxidase was also observed within acinar lumina. This work indicates that the predominant pathway taken by the HRP was via intercellular spaces and it is suggested that the permeability between junctional complexes of parenchymal cells is capable of being modified in vivo.


Effects of secretory nerve stimulation on acid phosphatase and  peroxidase in submandibular saliva and acini in cats.

The effects of parasympathetic or sympathetic nerve stimulation either alone, or in combination, on the acid phosphatase-containing central acinar cells and the peroxidase-containing demilunar cells of the cat submandibular salivary gland have been investigated by histochemical and cytochemical techniques. The results obtained with these techniques were correlated with biochemical assays for both enzymes in the saliva secreted. The results indicate that, although both sets of nerves probably affect both sets of cells, the predominant secretory effect of parasympathetic stimulation is on the central cells and, conversely, the predominant secretory effect of sympathetic stimulation is on the demilunes. Sympathetic stimulation appeared also to have initiated synthesis of peroxidase in the demilunar cells, especially when it was superimposed upon parasympathetic stimulation.


Secretory behaviour of hypertrophic and hyperplastic salivary gland.

The enzyme content and the secretory behaviour of normal rat salivary glands were compared with these properties in glands made hypertrophic and hyperplastic by the chronic administration of isoproterenol. The enlarged glands displayed reductions in the concentrations of ribonuclease, deoxyribonuclease and amylase. The secretory behaviour in vivo was similar for all enzymes in both types of glands, but the enlarged glands secreted a lower percentage of their content in vitro. The reduction in amylase activity was shown by immunological techniques to be due to a reduction in the number of enzyme molecules. The reduction in ribonuclease activity was not due to changes in the level of ribonuclease inhibitors.


Histochemistry and ultrastructure of amphibian lingual glands and phylogenetic relations.

The lingual glands of amphibians are confined to the dorsal face of the tongue and are formed by invaginations of the lingual epithelium. The secretory products have a heterogeneous composition. Mucosubstances are usually associated with proteins. The secretory product of the outer glandular cells is rich in mucosubstances of an acidity varying with species. In Anourans and Urodeles, the secretory product of the tubes contains abundant proteins and, where present, mucosubstances are less abundant and less acid than those produced at the surface of the tongue. Proteins and mucosubstances coexist in the same secretory granules, which exhibit a more or less homogeneous appearance in Gymnophiona and in Anourans while having a complex structure in Urodeles. In spite of their primitive anatomical features, the amphibian lingual glands present histochemical signs of a complete cellular evolution.


Vascular reactions to horseradish peroxidase in the guinea pig.

Albino guinea pigs were given intradermal injections of the protein tracer horseradish peroxidase. In a 0.1 mM concentration the tracer did not increase vascular permeability to Evans blue-labelled plasma proteins. In a 1 mM concentration, however, the peroxidase induced a local vascular leakage. This leakage was almost totally inhibited by pretreating the animals with acetylsalicylic acid, while antihistamine had only a weak inhibitory effect. We therefore believe that prostaglandins are important mediators in this HRP-induced vascular reaction.


Ultracytochemical localisation and comparison of adenyl cyclase activities in pineal bodies of Wistar rats and Mongolian gerbils.

In the pineal bodies of Wistar rats and mongolian gerbils (Meriones unguiculati) the main concentration of adenyl cyclase was found in capillary endothelial cells, their basal laminae, pericapillary amorphous material and in nerve endings. In the intercellular clefts between the rat pineal cells there was no adenyl cyclase activity, but a considerable amount of this enzyme was detected in the gerbils. The adenyl cyclase lacks completely in the pinealocyte cytoplasms of both species. It was concluded that the gerbil pineal cells may be more reactive to various hormonal influence than those of the rat.


A perfusion method of incubation to demonstrate horseradish peroxidase in bone.

A perfusion method of incubation to show horseradish peroxidase in the bone of young mice is presented. After perfusion fixation, the incubation medium is perfused from the descending aorta into the entire lower half of the animal. From the vessels there is good penetration of the medium into all tissues. This allows the preparation of any one perfused bone to ground-, semithin-, and ultrathin sections. Differences in peroxidase distribution in the entire bone suggest regional differences in vascular supply. The tracer enzyme diffuses freely from the vessels into the extracellular fluid of bone. 3 min. after injection, peroxidase is found between all bone lining cells and in osteocyte lacunae.


DNA-fluorometry of mammalian sperm.

The Dna-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescene distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.


Phenidone-ascorbic acid development in electronmicroscopic autoradiography.

Phenidone-ascorbic acid development in electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold section of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by cross-sectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible. Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.


Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy.

Cultured HeLa cells or mouse liver and pancreas tissues were labeled with 3H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes. After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and dry-mounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.


[The treatment of acute acoustic trauma (blast injury) with dextran 40 (author's transl)].

In a prospective study the results of treatment of 72 patients with deafness due to blast injury were assessed. All patients received infusions of Dextran 40. When treatment started within 3 days of the injury 74% showed complete recovery of hearing and the remaining 26% showed some improvement. When treatment started within 3 weeks of the injury 26.5% showed complete recovery, 61.8% had some improvement and 11.8% had no improvement. In 87.5% of those patients who attended after a longer interval no change in the audiograms occurred and only 12.5% had any significant improvement. The disappearance of tinnitus was effectively more common with early treatment. On account of these results the use of Dextran 40 is strongly advised.


[The reaction time as a source of error in threshold audiometry (author's transl)].

An investigation of reaction times at the threshold of hearing is described. 96 subjects were classified into two groups of sensorineural hearing loss utilizing Types I and II Bekesy audiometry, and compared with one normal group as control. The results show that reaction times are largely unaffected by the type of hearing loss and test frequency. Increasing age, however, may result in increasing reaction times. These results and possible sources of error are discussed as effecting an exact threshold audiometry.


[Diagnostic and therapeutic applications of bronchoscopy (author's transl)].

The development of therapeutic bronchoscopy into diagnostic bronchoscopy is described. Based on a personal experience of more than 35,000 bronchoscopies, attention is given to some important points in diagnosis and therapy. In diagnosis, these factors include: 1) color of mucosa, 2) structure of cartilage, 3) minimal tissue changes (nodules, vessels, folds), 4) tonus, 5) secretion, 6) miscellaneous peculiarities. Therapeutic studies involve: 1) recanalisation (from secretion to foreign body and tumor), 2) scrubbing (in treatment of fibrinous bronchitis and tracheitis, 3) bougies, 4) irrigation, 5) washing (for status asthmaticus, aspiration of gastric contents, etc.), 6) tamponade for persistent hemoptysis.


[Inhalation anaesthesia in bronchoscopies on infants and small children (author's transl)].

Over the past 5 years 37 bronchoscopies were performed on children aged 7 months to 3 years for the removal of foreign bodies, the diagnosis of inflammations, and the cause of chronic stridor. Because of improved instrumentation, illumination with "cold light" and cooperation between endoscopist and anaesthesist there were no complications. Anaesthesia was achieved by a mixture of oxygen, nitrous oxide and fluothane without use of muscle relaxants. Each child was given Atropine and Solu-Decortin H and if indicated Calcium gluconate and Euphillin. These endoscopies, except in three children with gross inflammation, were carried out as out-patient procedures. Early endoscopy in the doubtful case is desirable, since later complications may make anaesthetic conditions hazardous.


Side branch and parallel vent effects in real ears and in acoustical and electrical models.

The purpose of the project was to demonstrate and explain the acoustic effects of side branch and parallel vents in individual real ears. It was shown that parallel and side branch vents produce similar low frequency filtering effects and vent-associated reactance resonances. However, when the input system incorporates a side branch vent the sound pressure produced in the ear canal at frequencies above the vent-associated resonance is less than that produced when the input system is unvented. This effect is not seen when the input system is associated with a parallel vent. Data obtained in the real ear canals were compared to analogous measurements made using an acoustical model (the Zwislocki coupler) and an electrical model designed to simulate the hearing aid receiver, input tubing, earmold, ear canal, and eardrum. Both models yielded data very similar to the real ear results. The advantages of each model in predicting the effects of individual earmold vents are discussed.


Brainstem evoked response (BER) audiometry at various stimulus presentation rates.

The utilization of the latency of the brainstem auditory evoked response to estimate the status of high frequency hearing is rapidly becoming a part of the audiologist's clinical armamentarium. Because it is important to utilize the most efficient rate of stimulus presentation, the present investigation was undertaken to provide some initial latency information for three commonly used stimulus rates. Brainstem auditory evoked responses were recorded from 22 adults for three click rates (13.3/sec, 33.3/sec, and 67/sec) at seven intensity levels (10 to 60 dB sensation level). It was shown that rate of click presentation markedly affects the intensity-latency functions for wave V. In clinical use, norms must be established for each stimulus rate used to maximize the accuracy of the test results.


Sound levels in a 2-cc cavity, a Zwislocki coupler, and occluded ear canals.

Probe-tube measurements of the differences in sound levels at three locations in ear canals were compared to the differences in levels measured at analogous positions in a Zwislocki coupler and a 2-cc cavity. The results support the recommendation of Sachs and Burkhard that probe tube measurements should not be made with the probe tube flush with the earmold tip and close to the sound inlet bore. In real ear canals the transfer functions to the eardrum presented by Bruel, Frederikson, and Rasmussen and by Studebaker agree well with each other but differ somewhat from the one used by Sachs and Burkhard. In agreement with Bruel et al., the data of this study reveal a plateau in the relationship between real ear and 2-cc cavity responses between about 1.6 and 4.0 kHz, the relative intensity level of which may depend upon residual ear canal volume.


Pure tone delayed auditory feedback. Effect of hearing loss on disruption of tapping performance.

Pure tone delayed auditory feedback audiometry was administered to 20 veterans with normal hearing at 1 kHz and a sensorineural hearing loss associated with noise exposure at 4 kHz. Absolute and relative time errors as well as pattern errors were analyzed for the group and for each individual. Mean data showed no difference between error measures for the two frequencies. However, individual data revealed that errors in the subjects' performance occurred at a level slightly closer to threshold at the frequency with the hearing loss (4 kHz) than at the frequency at which hearing was normal (1 kHz).


Effects of human middle ear muscle contractions on speech intelligibility.

The present study assessed the relationship between the acoustic reflex and speech intelligibility. Monosyllabic words mixed with noise were presented at -6, -3.0 and +3 dB signal-to-noise ratios. The word lists were presented with a 2000-Hz tone in the contralateral ear at a level 15 dB above or 20 dB below the acoustic reflex threshold to evaluate intelligibility differences with the acoustic reflex contracted and relaxed. The results indicated that a significant decrement in speech intelligibility differences with the acoustic reflex contracted and relaxed. The results indicated that a significant decrement in speech intelligibility occurred with the reflex contracted at signal-to-noise ratios of -3, and 0 dB. Slight but not significant decrements were seen at -6 and +3 dB signal-to-noise ratios. These results are discussed along with possible reasons for the lack of agreement with previous research.


The mediastinum and hemothorax, pyothorax, and pneumothorax in the dog.

Contrary to the reported clinical, anatomic, and histologic evidence of communication between the 2 pleural cavities through the mediastinum in the dog, it was found that acute or chronic pathologic processes in one pleural cavity remained confined to that cavity unless the mediastinum was mechanically injured. These results were obtained in a series of experiments involving 39 dogs in which hemothorax or pyothorax or pneumothorax was unintentionally induced.


Bone conduction speech audiometry in normal subjects.

The present study was designed to investigate: (1) the relationship among bone conduction (BC) pure tone averages, BC speech reception thresholds (SRTs), and BC speech detection thresholds for normal subjects; (2) short term reliability of BC SRTs; and (3) characteristics of the articulation functions for spondees obtained by bone conduction. Twenty-five normal-hearing young adults participated. The data revealed that BC SRT-pure tone average and SRT-speech detection threshold relationships are essentially the same as for air conduction. A comparison of the articulation functions for air conduction and BC revealed no practical difference between the two modes of stimulus presentation.


Decibel notation with correlated and uncorrelated signals.

Audiologists perform sound level measurements in a variety of settings. These measurements typically involve clinical audiometric calibration, specification of sound field levels in industrial environments, psychoacoustic experimentation, or specification of sound levels in hearing and test boxes or anechoic chambers. Any of these circumstances can present problem situations in which some form of signal combination is involved. Solutions of such problems require use of the concepts of average power and signal correlation, as they are related to decibel notation. These concepts are discussed in the present paper, and several examples that apply these concepts to specific measurement situations are presented.


Auditory effects of noise on infant and adult guinea pigs.

This pilot study compared the susceptibility of the infant (48 hr) and adult (120 days) guinea pig to the effects of noise. Subjects were exposed to a narrow band of noise (center frequency 4 kHz) at an intensity of 115 dB sound pressure level (SPL) for 1 hr. Postexposure thresholds were obtained by a conditioned suppression technique. Results indicated that the infant animals displayed a mean hearing threshold of 25 dB SPL that significantly differed from the adult mean threshold of 7.5 dB SPL.


Septic polyarthritis associated with bacterial endocarditis in two dogs.

Two dogs were examined because of chronic shifting lameness. In each case, lameness was attributed to septic polyarthritis, as documented by synovial fluid analysis and culture. In 1 dog, antemortem diagnosis of bacterial endocarditis was verified by clinical and culture data. Treatment of both dogs was unsuccessful, and necropsy of each dog revealed bacterial endocarditis with coexistent septic polyarthritis. Bacteriologic blood cultures yielded an anaerobic Streptococcus sp (dog 1) and Pasteurella multocida (dog 2).


Myxomatosis in domestic rabbits in Oregon.

An epizootic of myxomatosis involved 26 rabbitries in western Oregon. Major clinical signs were inflammation and edema of the eyelids, conjunctiva, and anogenital area. Mortality ranged from 20 to 50%. On histologic examination, intracytoplasmic inclusion bodies were readily apparent in the epithelial cells of the conjunctiva. Lymphoid depletion of the spleen was also a common finding.


Eye closure and the acoustic reflex threshold.

Eye closure during measurement of the acoustic reflex threshold has been found on average to lower the threshold by 1.2 dB. It had no significant effect on the intra- or intersubject variability of the measure. It is concluded that it probably has little significance from the standpoint of clinical testing.


Admittance tympanometry in otosclerotic ears.

Twenty-eight surgically confirmed otosclerotic ears were evaluated to determine whether tympanometric admittance measurements could differentiate otosclerosis from a normal population. Subjects were tympanometrically measured at test conditions B220, B660, G220 and G660 Hz. When comparing mean curve peak amplitude to normative standards, 20 ears showed significantly low admittance tympanograms. Further investigation revealed no significant differences in curve width or pressure of the curve peak. Of the remaining eight otosclerotic ears, all produced diphasic "W"-notched tympanometric configurations. Diagnostic implications and physiological hypothesis are discussed.


Oak poisoning of cattle in Ohio.

Fourteen cases of oak poisoning were diagnosed at the Ohio Veterinary Diagnostic Laboratory, Reynoldsburg, Oh, in the fall of 1976. The poisoning was attributed to ingestion of oak shrubs, oak leaves, and acorns. Clinical signs included anorexia, rumen atony, hemorrhagic diarrhea, subcutaneous edema, and abnormal renal function. Perirenal edema and hemorrhage, ascites, hydrothorax, and hemorrhagic enteritis were frequently encountered pathologic changes. Histologic examination of the kidney revealed multifocal necrosis of the proximal convoluted tubules, which is a characteristic feature of this type of poisoning.


Plasma total protein measurement for prediction of disease and mortality in calves.

Plasma concentration of total protein (TP) was measured by refractometer in 76 calves, 1 to 8 days of age, on 3 farms. Three calves died before weaning at 5 weeks; all had plasma TP less than 6 g/dl. The frequency of disease before weaning was 17/29 (59%) in calves that had plasma TP of less than or equal to 6 g/dl, and this was significantly (P less than 0.001) less than the disease frequency of 9/47 (19%) in those with higher values of plasma TP. For 9 calves, plasma TP content was 4.72 +/- 0.22 (mean +/- SD) before ingestion of colostrum. The increment of plasma TP after colostrum feeding was 2.03 +/- 0.61 mg/dl (P less than 0.001). Plasma TP was as useful as serum zinc sulfate turbidity test results or total immunoglobins (as measured by radial immunodiffusion) in predicting disease susceptibility during the first 5 weeks of life, based on determinations made in 32 calves during the first 8 days of life. It was concluded that determination of plasma TP is the most convenient method of evaluating the immune status of calves under field conditions.


Haemophilus parasuis infection in swine.

Septicemic disease occurred in 49 of 126 pigs several days after being transported 80 km. All affected pigs died. The main changes in acutely affected pigs were skin discoloration, pulmonary edema, arthritis, meningitis, and renal glomerular thrombosis. In peracute cases, gross findings were minimal. Haemophilus parasuis was isolated from multiple organ sites in most affected pigs. Haemophilus parasuis was isolated from nasal swab specimens from 17 of 20 clinically normal pigs on the farm of origin. Fatal acute septicemia was reproduced in 2 pigs by intravenous or intratracheal exposure to an isolant of H parasuis obtained from 1 of of the 49 fatally affected pigs. Aerosol exposure of 5 pigs resulted in mild pneumonia in 4 pigs and severe pneumonia, pleurisy, pericarditis, and terminal septicemia in 1 pig.


Intramural esophageal cyst in a horse.

A yearling Quarter Horse colt was examined because of intermittent esophageal obstruction. A soft tissue mass was palpated in the cervical portion of the esophagus. Plain and contrast radiography and esophagoscopy were utilized to confirm the location of the lesion in the esophageal wall. Surgical exploration and removal of the mass was performed through a ventral midline cervical incision. Histologically, the mass was a keratinizing squamous epithelial inclusion cyst.


The effect of adrenal glucocorticoid administration on eruption rates and tissue dimensions in rat mandibular incisors.

A quantitative investigation was undertaken to evaluate the changes which occur in the dental tissues of the rat incisor following the systemic administration of adrenal glucocorticoid hormone. Of the 53 rats used in the 5 week experiment, 21 acted as untreated controls while the remaining 32 each received weekly subcutaneous injections of 0-1 ml Depo-Medrone. Throughout the experimental period the mandibular incisor eruption rates of a selection of both groups of rats were recorded. Transverse ground sections were prepared from the left mandibular incisor of each animal. Measurements taken on sections prepared at a standard reference level enabled comparisons to be made between control and hormone-treated animals in the dimensions of their incisor dentine and pulp. The results indicated that maintaining mature rats in a hyperglucocorticoid state caused a significant increase in incisal eruption rate, together with a thinning of the dentine walls and an increase in size of the pulp chambers of these teeth.


Remodelling in immature hyaline cartilage: changes in matrix dry mass.

Scanning microinterferometric measurements were made of matrix dry mass in the intermediate layer of normal rabbit patellar articular cartilage and in the area surrounding standard, experimental, full-thickness lesions of 1-5 mm diameter. The lesions were created in 1 kg animals and were allowed to heal for up to 3 weeks. Changes in matrix dry mass were recorded in predetermined zones around the lesions immediately after surgery, and at 1 week, and at 3 weeks post-operatively. After 1 week the rapid early fall in matrix solids was partly offset in areas corresponding to a zone of locally proliferating chondrocytes which appeared to maintain the matrix dry mass close to normal levels. The maximal fall occurred at the extreme edge of the lesion where, it is suggested, total glycosaminoglycan loss had taken place. After 3 weeks the matrix dry mass values had risen to the normal range, apart from a narrow zone adjacent to the wound edge. The findings are discussed in the light of previous quantitative work on the composition of articular cartilage and on the nature of the reactions of cartilage to injury.


Cortical bone loss in triamcinolone-treated mice.

Immature mice were treated for up to 8 weeks with daily doses of triamcinolone diacetate. The humerus was studied radiographically at regular intervals. Roentgenographic measurements included midshaft width, medullary width, cortical thickness and total length. By the time of the tenth injection significant cortical bone loss was noted, followed by increased width of the medullary cavity and osteoporosis. An initial phase of active endosteal bone resorption was replaced by a prolonged steady state which extended throughout the experimental period. Possible explanations for the above findings are discussed.


Changes in the mucosal surface area of the small gut of rats of different ages.

Changes in the mucosal surface area of the small intestine of the ageing male Wistar rat were investigated. The results show that the mucosal surface area (Ma) and, even more, the mucosal area per unit serosal area (Ra) diminishes along the length of the small intestine (excluding the duodenum) in the young rat. In adults, however, there is relative constancy of these parameters along the small intestine. The serosal circumference (Sc) of the intestinal segments investigated increased considerably with advancing age.


Histochemistry of blood and bone marrow smears of the straw-coloured fruit-eating bat, Eidolon helvum.

Blood and bone marrow cells of the straw-coloured fruit bat, Eidolon helvum were examined for Sudan black B and periodic acid-Schiff (PAS)-positive material, peroxidase, naphthol ASD chloroacetate esterase and beta-glucuronidase. A strong peroxidase reaction was given by developing granulocytes and monocytes in bone marrow smears, but the mature cells in the peripheral blood smears showed no reactivity. Further, developing and mature erythrocytes in both blood and bone marrow smears showed intense peroxidase reactivity.


A quantitative study of myelination following hypoxic stress.

The total number of axons, percentage myelination, mean number of myelin lamellae, and mean diameters of myelinated and unmyelinated axons, were studied in the anterior limb of the anterior commissure in mice subjected to hypoxia at 390 mm Hg for two days. There was no significant difference in any of these parameters, either between the control group and the hypoxic group, or between the control group and a group allowed to recover for one week after removal from the decompression chamber. This suggests that the astrocytes, which decrease in number markedly following hypoxia, are not necessary for the metabolic support of the myelin sheath, either directly or indirectly via an influence on the oligodendrocytes.


Uptake of horseradish peroxidase by sensory nerve fibres in vitro.

After 47 3/4 hours in vitro, organotypic cultures of dorsal root ganglia of rat were exposed to horseradish peroxidase at a concentration of 4 mg/ml of nutrient medium for 15 minutes. Observations on the living nerve fibres revealed pinocytotic vesicles in the growth cones which were transported toward the perikarya for several hundreds of micrometer at a rate of 0-5-10 micrometer/min. Electron microscopic sections displayed uptake of peroxidase into the growth cones by means of endocytosis. The enzyme marker was contained in small-sized vesicles, as well as in large vacuoles which measured up to 2 micrometer in diameter. The more proximal parts of the axon contained tiny vesicles which were well below the level of resolution of the light microscope. Pinocytotic phenomena were abundant at the fibre tips, but could also be observed occasionally in the intermediate and more proximal portions of contiguous axons. It is proposed tentatively that the large vacuoles in the distal and intermediate parts of the axon had formed from the small vesicles. Furthermore, it is suggested that the profuse uptake and backward transport of vesicles in these regenerating neurons might be meaningful for the neuron in terms of intercellular and environmental recognition.


Mechanisms by which histamine stimulates rapidly adapting receptors in dog lungs.

The stimulant effect of histamine on intrapulmonary rapidly adapting receptors was studied in anesthetized, paralyzed, artificially ventilated dogs, to determine whether it was dependent on contraction of airway smooth muscle. Single- or few-afferent fiber recordings were made from small strands dissected from the otherwise intact vagus nerve. Aerosols of histamine increased both receptor discharge and tracheal pressure (Pt). In 27 experiments, isoproterenol prevented the increase in Pt but not in receptor activity induced by histamine. In another 32 experiments, acetylcholine (ACh) increased Pt to a greater extent than did histamine, whereas histamine increased receptor discharge more than ACh. When given as aerosol directly to the vicinity of the sensory receptor via a fiber bronchoscope, histamine stimulated 12 receptors, whereas ACh and buffer solution did not. Isoproterenol reduced slightly the stimulant effect of locally applied histamine on 8 receptors. We conclude that smooth muscle contraction is not essential for stimulant effects of histamine on rapidly adapting receptors in dog lungs, and that histamine acts directly on the receptors to increase their activity.


Influence of exercise and heat stress on pulmonary function during ozone exposure.

We studied the effects of a 2-h exposure to ozone (0.5 ppm) in 14 nonsmoking males under four environmental conditions (WBGT (wet bulb-globe temperature index) = 64.4, 80.0, 85.2, and 92.0 degrees F). The subjects were divided into two groups, A (n = 8) and B (n = 6). Thirty minutes of exercise at 40% Vo2 max was performed from 60 to 90 min for group A and 30 to 60 min for group B. Pulmonary function changes, determined throughout exposure, were greatest immediately after exercise in both groups. Few changes occurred before the exercise period. However, the decrease in FVC (826 ml) and FEV1.0 (937 ml) following exercise was more than twice as large as the decrement seen at end exosure (388 and 423 ml, respectively). Measures of maximum expiratory flow (FEF 25-75%, FEF 50% FEF 75%) showed similar reductions. In many cases, reversal of these changes occurred during the remainder of the exposure period. The greatest decrease in FVC occurred when heat and ozone exposures were combined (WBGT = 92.0 degrees F). We conclude that the effects of ozone are most severe immediately after exercise and that heat stress may modify the overall effect of ozone on pulmonary function.


In vivo gas trapping induced by nitrous oxide.

Repeated vital capacity (VC) breaths in 17 anesthetized and curarized dogs induced a small but significant increase in residual volume (RV). This trapping was greatly enhanced when a mixture of 80% N2O and 20% O2 (N2O-O2) was used instead of air. VC breaths with N2O-O2 also increased closing volume, closing capacity and closing pressure determined with 133Xe. Successive quasi-static pressure volume (PV) curves of the lung showed a shift to the right at high lung volume and to the left at low lung volume. Again these changes were more impressive with N2O-O2). The PV curve of the chest wall was unchanged. Insufflations from RV were necessary to produce the gas trapping. Increase in RV was positively related to the size of the inspired volume, to N2O concentration, and inversely related to the inspiratory flow rate. Vagotomy, intravenous isoproterenol, and intravenous propanolol did not alter the phenomena. We propose that these observations can be explained by the presence of foam in the airways and that N2O leads to an increase of foam by osmotic liquid shift into the bronchioli, and/or to an increase in bubble's size by gas diffusion.


Renin, aldosterone, electrolyte, and cortisol responses to hypoxic decompression.

Responses of plasma renin activity, plasma aldosterone, plasma cortisol, and plasma electrolyte concentration and urinary electrolyte and aldosterone excretion were studied in four men during hypoxic decompression to a stimulated altitude of 4,760 m in a pressure chamber. Three of the four subjects developed significant acute mountain sickness. Plasma sodium and potassium concentrations were unchanged. No significant change in plasma renin activity was observed, but values tended to fall. Plasma aldosterone concentration was depressed while plasma cortisol was elevated and diurnal variation lost. Urinary sodium excretion was unchanged, but urinary potassium and aldosterone excretion were decreased. The decrease in plasma and urinary aldosterone and urinary potassium in the absence of change in plasma renin activity or plasma potassium is of uncertain origin. It is unlikely to be due to a decrease in adrenocorticotropin secretion since plasma cortisol rose during the same time. None of the changes could be causally implicated in the development of acute mountain sickness although the increase in plasma cortisol was greatest in the most ill.


Muscular efficiency during steady-rate exercise. II. Effects of walking speed and work rate.

A comparison of walking against vertical (gradient) and horizontal (trailing weight) forces was made during steady-rate exercise at 0.250, 500, and 750 kg-m/min with speeds of 3,0, 4.5, and 6.0 km/h. In all cases exponential relationships between energy expenditure (calculated from the steady-rate respiration) and increasing work rate and speed were observed which indicated that muscular efficiency during walking is inversely related to speed and work rate. "Work" (level, unloaded walking as the baseline correction), "delta" (measured work rate as the baseline correction), and "instantaneous" (derived from the equation describing the caloric cost of work) efficiencies were computed. All definitions yielded decreasing efficiencies with increasing work rates. At work rates above 250 kg-m/min the curves describing the relationship between energy expenditure and work rate were parallel for vertical and horizontal forces, indicating equivalent efficiencies in this range. Only the delta and instantaneous definitions accurately described these relationships for vertical and horizontal work. Determinations of combined work loads (gradient plus trailing weight) were made and the energy costs of both types of work found to be additive.


Temperature and CO2 effect on phrenic activity and tracheal occlusion pressure.

The present investigation was undertaken to study the interaction of CO2 and body temperature on phrenic activity (moving average) and tracheal occlusion pressure. Studies were performed on spontaneously ventilated cats anesthetized with pentobarbital sodium at different body temperatures (32-41 degrees C) while breathing room air, 2 and 4% CO2 in 50% O2. At any given chemical drive, increased body temperature caused a similar increase in rate of phrenic activity and tracheal occlusion pressure, while their peak values remained virtually unchanged. At any given body temperature, increased chemical drive caused an increase in both rate of rise and peak values of phrenic activity and tracheal occlusion pressure. These results confirm previous findings that body temperature affects the rate of rise of the central inspiratory activity (CIA), but not the inspiratory "off-switch" threshold, while CO2 increases both the rate of rise of CIA and off-switch threshold. In addition the results indicate that tracheal occlusion pressure provides a similar index of CIA as "integrated" phrenic activity.


Protein metabolism by rat lung: influence of fasting, glucose, and insulin.

We studied protein metabolism by rat lung slices. We found that phenylalanine is not metabolized to other substances by the lung and that the rate of incorporation of L-[U-14C]phenylalanine into protein, calculated using its intracellular specific radioactivity, reached a maximum within 20 min and remained stable for the rest of a 3-h incubation. The rate of protein degradation, determined using [12C]phenylalanine as a marker, was linear over a 3-h incubation. Fasting for 3 days slowed the increase in lung protein content of fasted compared to nonfasted rats; there was also a decrease in protein synthesis and an increase in proteolysis. In fed rats, glucose, insulin, and glucose plus insulin did not alter protein synthesis. Glucose, insulin alone, and glucose plus insulin decreased proteolysis. We conclude that the in vitro system reflected changes in the in vivo protein content of the lung. Fasting decreases protein synthesis and increases proteolysis. Glucose and insulin alone modulate protein metabolism in the lung by acting on the degradative rather than the synthetic process.


Respiratory patterns during progressive asphyxia in newborn rabbits.

During acute progressive asphyxia an abrupt transition occurs from regular respiratory efforts to primary apnea and gasping. To study this transition we measured inspiratory duration (TI), expiratory duration (TE), maximal inspiratory tracheal pressure (Pmax), electromyographic activity (EMG) of different muscle groups, and the electrocorticogram (ECoG) of 3- to 5-day-old rabbits following airway occlusion at functional residual capacity (FRC). The onset of primary apnea was associated with other central nervous system events as indicated by decerebrate posture and maximal EMG activity which were followed by relaxation, loss of EMG activity, and an isoelectric ECoG. Indices of inspiratory drive (Pmax and Pmax/TI) were preserved or accentuated following primary apnea despite a reduction of overall respiratory activity (frequency X Pmax). The onset of primary apnea appears to be due to interruption of the respiratory cycling controls rather than failure of the respiratory center which regulates inspiratory force. Gasping appears to be linked with progressive changes in respiratory patterns which are observed prior to primary apnea. These findings do not support the concept of a "gasping" center.


Quantitative analysis of the front crawl in men and women.

Body drag, D, and the overall mechanical efficiency of swimming, e, were measured from the relationship between extra oxygen consumption and extra drag loads in 42 male and 22 female competitive swimmers using the front crawl at speeds ranging from 0.4 to 1.2 m/s. D increased from 3.4 (1.9) kg at 0.5 m/s to 8.2 (7.0) kg at 1.2 m/s, with D of women (in brackets) being significantly less (P less than 0.05) than that of men. Mechanical efficiency increased from 2.9% at 0.5 m/s to 7.4% at 1.2 m/s for men, the values for women being somewhat greater than those for men. The ratio, D/e was shown to be identical to the directly measured energy cost of swimming one unit distance, V02/d, and was independent of the velocity up to 1.2 m/s. It averaged 52 and 37 l/km for men and women respectively (P less than 0.05). When corrected for body surface area the values were 27 and 22 l/km-m2 for men and women, respectively (P less than 0.05). The underwater torque, T, a measure of the tendency of the feet to sink, was 1.44 kg-m for men and 0.70 kg-m for women (P less than 0.05). VO2/d increased linearly with T for both men and women of similar competitive experience. However, the proportionality constant delta VO2/d-delta T was significantly less for competitive than noncompetitive swimmers. The analysis of the relationship VO2/d vs. T provides a valuable approach to the understanding of the energetics of swimming.


Albumin clearance from alveoli: tissue permeation vs. airway displacement.

In anesthetized, ventilated dogs, clearance of 2 +/- 0.2 ml alveolar-instilled 1% isosmolar [125I]albumin ([125I]RISA) was separated into bulk airway displacement and transalveolar tissue permeation by the use of collimated external detectors and by sampling of blood and lymph. The detectors were positioned perpendicular to the plane of bronchial drainage, and collimator resolution was characterized by a 50% decrease in count rate for a 5-mm lateral (transbronchial) instillate shift. Detector signals demonstrated no airway shift in 10 dogs studied, the signal decay equaling [125I]RISA absorption determined by lung homogenates. The mean rate constants of [125I]RISA lung clearance and epithelial permeation were 4.08 X 10(-4)-min-1 and 5.48 X 10(-4)-min-1, respectively. The diffusional permeability coefficient of alveolar epithelium for albumin was 4.06 X 10(-9) CM-S-1. Absorbed [125I]RISA was separated into blood and lymph components by collecting lymph from the right lymphatic duct (RLD) and thoracic duct. Mean blood/lymph removal ratio was 6.3/1, and correlated with plasma/RLD lymph steady-state albumin concentration ratios in individual animals. Over a mean observation period of 5.4 h, an average of 12.3% [125I]RISA was cleared from the instilled region. The data suggest that this amount was cleared by permeation; there was little evidence of airway clearance.


Wave-speed limitation on expiratory flow-a unifying concept.

The mechanism limiting forced expiratory flow is explained on the basis that a local flow velocity reaches the local speed of wave propagation at a point, called the choke point, in intrathoracic airways. This theoretical approach to the "waterfall effect" leads to selection of the analogy of constricted open-channel flow to apply to the elastic network of airway tubes. Quantitative results are derived for the case of negligible friction by use of the Bernoulli principle. Shapes predicted for the maximum-flow static recoil curves depend only upon the nature of the pressure-area curve at the choke point in the case of negligible friction; and the magnitude of the critical rate of flow depends on reference values of cross-sectional area and elastic modulus at the choke point, on gas density, and on the static recoil pressure. The present theoretical results are used to interpret previous experiments, but quantitative applicability is limited because of frictional effects and lack of knowledge of choke point conditions.


Airway geometry by analysis of acoustic pulse response measurements.

Serial distribution of airway properties determines in part the response of the lung to high frequency oscillations. We measured the response of excised dog lungs and lobes between 156 and 10,000 Hz and determined the area-distance function of the acoustically equivalent structure having rigid walls, regular branching, and negligible internal losses. The utility of this techique was tested by determining the effects of air trapping, removal of pleura from a dried lung, central airway smooth muscle tone. A strong correlation was found between relative changes in equivalent acoustic area and relative area changes measured radiographically in individual airways at corresponding distances. We conclude that despite departures of the properties of the real lung from the characteristics of the acoustically equivalent structure, changes in the area-distance function computed by this technique provide reasonable estimates of the magnitude and serial distribution of actual changes in airway cross-sectional area.


Maximum expiratory flow changes induced by longitudinal tension on trachea in normal subjects.

Maximal expiratory flow (Vmax) was noticed to increase in some subjects during neck hypertension. Maximal expiratory flow volume (MEFV) curves were obtained in 15 normal young subjects at regular and hyperextended neck posture. Eleven of the subjects had consistently higher Vmax during neck hyperextension at high lung volumes, accompanied by MEFV configuration changes in the form of the obliteration of a concavity towards the volume axis that existed in the curve at regular neck posture. Effort independency was documented at lung volumes where the changes occurred. Radiographic studies indicate tracheal elongation with a relatively fixed carina during neck hyperextension. We propose that at high lung volumes in normal young subjects, the flow-limiting mechanism resides in the trachea and that the increased Vmax with neck hyperextension. We propose that at high lung volumes in normal young subjects, the flow-limiting mechanism resides in the trachea and that the increased Vmax with neck hyperextension reflects the effect of tracheal elongation which stiffens the trachea under dynamic conditions and increases its tube-wave speed. This concept was confirmed by MEFV curves obtained from anesthetized tracheostomized dogs when increased tracheal longitudinal tension resulted in an increase of Vmax.


A digital computer technique for analyzing respiratory muscle EMG's.

A method is described for extracting from the electromyograms of respiratory muscles a continuous signal which has primarily the periodicity of respiratory pressure and flow wave forms. The EMG is first band-pass-filtered from 50 to 500 Hz, then digitized, full-wave rectified, passed through a nonlinear voltage window to reduce noise (particularly ECG) artifacts, then low-pass filtered with a digital continuous, or moving, averager. An average wave form corresponding to one respiratory cycle is produced by ensemble averaging of the wave forms from several consecutive breaths. Diaphragmatic electromyograms from a human and from a rabbit are processed in this manner, and the effect on the processed wave forms of changes in inspired CO2 and of a change in end-expiratory lung volume are demonstrated.


An analog approach to measuring O2 uptake on-line.

An analog circuit has been designed to provide a solution to the Haldane transformation that yields true O2 fraction. Incoming signals proportional to fractional concentration of CO2 and O2 in mixed expired gas are utilized in the circuit and the results are multiplied by a value proportional to ventilation. The final products are analog signals proportional to O2 uptake and CO2 output. The system is applicable to the anesthetized and paralyzed animal kept at a known constant ventilation. The final products are analog signals proportional to O2 uptake and CO2 output. The system is applicable to the anesthetized and paralyzed animal kept at a known constant ventilation. Diagrams of the circuit and expired gas collection and analysis arrangements are provided.


Panic-fear in asthma: generalized personality trait vs. specific situational state.

Previous studies have found that both ASC and MMPI Panic-Fear scale scores are related to the maintenance of the medical intractability of asthma. Despite the large overlap of these two scales, they measure somewhat different aspects of "Panic-Fear". The ASC measures a situational response (state variable) to breathing difficulties, while the MMPI measures a more stable, diffuse, and global personality characteristic (trait variable). The present study analyzes the relationship between these state and trait variables as they relate to the intensity of prescribed discharge medication. The results indicate that it is the trait variable which is more important in the maintenance of the medical intractability of asthma. The state variable is implicated in maintaining the illness only to the extent that it is a specific manifestation of the trait variable.


Bisulfite reductase of Desulfovibrio vulgaris: explanation for product formation.

Bisulfite reductase, purified from Desulfovibrio vulgaris, was coupled with the pyruvate phosphoroclastic reaction. Moderate to low reducing conditions resulted in the formation of trithionate; however, when the concentration of reductant was high, a mixture of trithionate and thiosulfate was formed. Sulfide was also a detectable product, but only when the concentration of bisulfite was low. Flavodoxin mediated native coupling between bisulfite reductase and the phosphoroclastic reaction. A model for bisulfite reductase activity is proposed.


Effect of urea concentration on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 x 10(4) colony-forming units per ml. The maximum yield of organisms, 8.0 x 10(7) colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 x 10(10) total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K(s) value for growth was 2.0 x 10(-4) M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.


Mercury and organomercurial resistances determined by plasmids in Staphylococcus aureus.

Penicillinase plasmids of Staphylococcus aureus often contain genes conferring resistance to inorganic mercury (Hg(2+)) and the organomercurial phenylmercury acetate. The mechanism of resistance was found to be the enzymatic hydrolysis of the organomercurial phenylmercury to benzene plus inorganic ionic mercury, which was then enzymatically reduced to metallic mercury (Hg(0)). The Hg(0) was rapidly volatilized from the medium into the atmosphere. After the mercurial was degraded and the mercury was volatilized, the resistant cells were able to grow. These plasmids also conferred the ability to volatilize mercury from thimerosal, although the plasmid-bearing strains were equally as thimerosal sensitive as the S. aureus without plasmids. None of the plasmids conferred the ability to volatilize mercury from several other organomercurials, however: methylmercury, ethylmercury, p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate. (Organomercurial resistance-conferring plasmids of Escherichia coli and Pseudomonas aeruginosa that we have been studying confer the ability to degrade two or three of these organomercurials.) Although mercury was not volatilized from p-hydroxymercuribenzoate or fluorescein mercuric acetate, the plasmid-bearing strains were resistant to these organomercurials. The ability to volatilize mercury from Hg(2+) and phenylmercury was inducible. The range of inducers included Hg(2+), phenylmercury, and several organomercurials that were not substrates for the degradation system. Mercury-sensitive mutants have been isolated from the parental plasmids pI258 and pII147. Thirty-one such mercury-sensitive strains fall into three classes: (i) mercury-sensitive strains totally devoid of the phenylmercury hydrolase and Hg(2+) reductase activities; (ii) mutants with normal hydrolase levels and no detectable reductase; and (iii) mutants with essentially normal hydrolase levels and low and variable (5 to 25%) levels of reductase activities. The mercury-sensitive strains were also sensitive to phenylmercury, including those with the potential for hydrolase activity.


Protein synthesis during morphogenesis of Mucor racemosus.

Cells of Mucor racemosus were labeled with l-[(14)C]leucine during the yeast-to-hyphae morphogenesis that follows a change of atmosphere from CO(2) to air. Pulse-labeling kinetics and the steady-state accumulation of incorporated l-[(14)C]leucine were determined throughout the period of cellular differentiation. We determined that the l-[(14)C]leucine was taken up by all forms of the organism, was not altered from the form of l-leucine, and was incorporated exclusively into protein. The intracellular pool of free l-leucine was small in comparison with those of the other l-amino acids, remained relatively constant in size during morphogenesis, and was rapidly equilibrated with exogenous leucine. Approximately the same internal radiospecific activities were attained throughout development shortly after addition of l-[(14)C]leucine to a culture. Experiments performed with leucine auxotrophs suggested that endogenous synthesis of leucine in prototrophs does not affect the measured rates of incorporation. Experiments performed with (14)C-labeled l-isoleucine, l-proline, l-lysine, and l-arginine produced results qualitatively the same as with l-leucine. The accumulation of incorporated l-[(14)C]leucine in a culture of M. racemosus undergoing the air-induced yeast-to-hyphae transition reflected the change in growth rate that accompanied the morphogenesis. However, the specific rate of protein synthesis measured throughout the developmental process displayed a characteristic acceleration during the emergence of germ tubes which was followed by a decline when all further growth took the form of hyphal elongation. Data are presented suggesting that this response is a correlate of morphogenesis rather than a consequence of the atmospheric change per se.


Membrane lipid biosynthesis in Acholeplasma laidlawii B: incorporation of exogenous fatty acids into membrane glyco- and phospholipids by growing cells.

The extent of incorporation of a wide variety of exogenous saturated, unsaturated, branched-chain, and cyclopropane fatty acids into the membrane lipids of Acholeplasma laidlawii B was systematically studied. Within each fatty acid class the extent of incorporation generally increased markedly with increasing chain length, reached a maximum, and then declined progressively but less sharply with further increases above that chain length giving maximal direct incorporation. Certain shorter-chain members of each fatty acid class underwent complete or partial conversion to longer-chain homologues before utilization for complex lipid biosynthesis. The degree and extent of chain elongation and direct incorporation and the characteristic dependence of each of these processes on fatty acid chain length and structure correlated well with the physical properties (melting temperatures) of the exogenous fatty acids. The in vivo specificity of the enzyme systems responsible for the incorporation of exogenous fatty acids was such that the fluidity and physical state of the membrane lipids were maintained within a definite, albeit a relatively wide, range. We also observed that the neutral glycolipids typically have similar fatty acid compositions, which are somewhat different from those of the major phosphatides, which also exhibit similar fatty acid spectra. The phosphorylated glycolipid glycerophosphoryldiglucosyl diglyceride, however, always maintained a unique fatty acid composition quite different from that of the diglucosyl diglyceride from which it is presumably derived. These characteristic differences in fatty acid composition appear to function to minimize differences in phase transition temperatures, thus producing a more physicochemically homogeneous mixture of membrane lipids than would result from a nonspecific incorporation of fatty acids.


Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells.

The de novo biosynthesis of fatty acids of 12 to 18 carbons from precursors of 5 carbons or fewer has been demonstrated in Acholeplasma laidlawii B. Radiolabeling experiments indicated that the normal primers for the synthesis of the even- and odd-chain fatty acids are acetate and propionate or valerate, respectively. Saturated straight-chain monomethyl-branched fatty acids of up to five carbons were readily utilized as primers, wheras more highly branched species and those possessing halogen substituents or unsaturation were not utilized. At primer concentrations of 1 to 3 mM, up to 80% of the total cellular lipid fatty acids were derived from exogenous primer. The mean chain length of the exogenous primer-derived fatty acids rose with increasing primer incorporation for methyl-branched short-chain fatty acids but was invariant for propionate. The products of de novo biosynthesis varied only slightly with temperature or cholesterol supplementation, suggesting that de novo biosynthesis is not directly influenced by membrane fluidity. Cerulenin inhibited de novo biosynthesis in a fashion that suggests the presence of two beta-ketoacyl thioester synthetases, which differ in substrate chain length specificity and in susceptibility to inhibition by the antibiotic.


Macromolecular synthesis and degradation in Arthrobacter during periods of nutrient deprivation.

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.


Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum.

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60 degrees C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; alpha-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V(max) and K(M) values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective alpha-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the alpha-ketoacid and CO(2). The data indicate that the two enzymes are similar to pyruvate synthase and alpha-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.


Purification and partial characterization of the principal deoxyribonucleic acid polymerase from Mycoplasmatales.

In this report we present the first description of the isolation and partial characterization of the deoxyribonucleic acid (DNA) polymerase activity from two species of Mycoplasmatales, Mycoplasma orale type 1 and M. hyorhinis. We have identified only a single DNA polymerase species in the mycoplasma crude extracts, and the enzymes from the two organisms are very similar in their structural and enzymatic properties. The purified polymerase from each source has a specific activity of greater than 50,000 U/mg of protein, a sedimentation coefficient of 5.6s, and an estimated molecular weight by gel filtration of 130,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the most highly purified M. orale fraction contains a single major protein band of 130,000 daltons, which we believe may represent the polymerase protein. The enzymes are most reactive with gapped (activated) DNA and show a marked preference for this primer template over oligodeoxyribonucleotide-initiated homoribo- or homodeoxyribo-polymers. The most purified preparations are devoid of contaminating endonuclease activity and also appear to lack associated 5' leads to 3'- or 3' leads to 5'-exonuclease activities, as determined by highly sensitive assays. The absence of the 3' leads to 5'-exonuclease is particularly remarkable in that this activity is essentially ubiquitous among the DNA polymerases that have thus far been characterized from procaryotes.


Molecular basis for the maximum growth temperature of an obligately psychrophilic yeast, Leucosporidium stokesii.

Cells of the obligately psychrophilic yeast Leucosporidium stokesii were subjected to permissive (15 and 20 degrees C) and restrictive (23 and 25 degrees C) temperatures to determine the event(s) responsible for the low maximum growth temperature of this organism. An investigation of subcellular morphology by nuclear staining revealed that buds formed at 20 degrees C were anucleate but showed nuclear migration within the parent cell. Cells incubated initially at 23 degrees C and then shifted down to a permissive growth temperature of 15 degrees C in the presence of a deoxyribonucleic acid (DNA) synthesis inhibitor, hydroxyurea, confirmed the observation that the anucleate condition of atypical buds was the result of temperature-sensitive DNA synthesis. Concomitantly, the incorporation of labeled adenine into DNA was inhibited at 23 and 25 degrees C. The synthesis of ribonucleic acid, however, was enhanced at 23 degrees C but impaired at 25 degres C. Similarly, protein synthesis was unaffected at either restrictive temperature.


Distribution and conformation of crystalline nigeran in hyphal walls of Aspergillus niger and Aspergillus awamori.

Hyphal walls of Aspergillus awamori containing increased amount of the alpha-glucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by x-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack.


Caulobacter flagellins.

Flagellins from 17 Caulobacter strains were partially purified. Their molecular weights and immunological properties indicated that a two-component flagellin system is common among Caulobacters.


Connectin, an elastic protein of muscle. Its abundance in cardiac myofibrils.

Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).


Mitochondrial sulfhydryl groups. A possible endogenous probe of conformational changes in the mitochondrial membrane.

The protein-bound sulfhydryl (SH) groups of the mitochondrial membrane were determined with Ellman's reagent in energized and non-energized configurational states of mitochondria and submitochondrial particles. When beef heart mitochondria were energized by respiration, there was a decrease in titratable protein-bound SH groups which varied according to substrate: NADH-linked substrates induced a decrease of about 10 nmol per mg of protein,succinate about 7, and ascorbate-tetramethyl-p-phenylene-diamine about 3. Similar changes occurred in phosphorylating submitochondrial particles. A decrease in SH titer was also observed in non-energized conditions, induced by hypotonic treatment and by some reagents inhibiting electron transport and oxidative phosphorylation and inducing orthodox configuration. These changes in protein-bound SH groups might be useful in analyzing the conformational states of mitochondrial membranes.


Purification and some properties of glutathione-S-epoxide transferase from guinea pig liver.

Glutathione-S-epoxide transferase has been purified from guinea pig liver in a homogeneous form having a sedimentation coefficient, s20,w of 3.8S, and a molecular weight of 46,000, and dissociable into subunits with a molecular weight of 25,000. The activities with four substrates, styrene oxide, naphthalene oxide, iodomethane, and p-nitrobenzyl chloride, were all copurified during purification of the enzyme from 100,000 X g supernatant of guinea pig liver. However, kinetic data suggest different mechanisms for the glutathione conjugating reaction with iodomethane and p-nitrobenzyl chloride. The view that the epoxide transferase does not distinguish between simple epoxides and arene oxides has been confirmed with the guinea pig liver enzyme.


Decrease of seminolipid content in the testes of rats with vitamin A deficiency determined by high performance liquid chromatography.

Changes of seminolipid content in vitamin A-deficient rat testes were quantitatively determined by improved and facile high performance liquid chromatography using a reversed phase column under an ion pair chromatographical condition. The seminolipid content of the testes of rats fed a vitamin A-deficient diet for 46 days decreased to 13% of that of the control rats fed a vitamin A-deficient for 20 days and then supplemented with 140 microgram/rat/day of vitamin A palmitate for 26 days. Total lipid, phospholipid and DNA of vitamin A-deficient rats were slightly reduced. Histological examinations showed that seminiferous tubular cells degenerated to aspermatogenesis with a vitamin A-deficient diet. The remarkable reduction of seminolipid content, compared to the slight decrease of DNA content, is considered to be the result of damage to the seminiferous tubular elements, especially at the differentiated step of germinal cells from spermatogonia to the next. These results further support the theory that seminolipid biosynthesis is characterized by strict dependence on the differentiation of seminiferous tubular cells or spermatogenesis.


Pea histones H2A and H2B. Variable and conserved regions in the sequences.

Pea histone II group, a mixture of H2A and H2B obtained by chromatography on an ion-exchange resin, was further fractionated by carboxymethylcellulose chromatography and purified by Bio-Gel P-60 chromatography. Their chromatographic behaviors and gel electrophoretic mobilities of single bands differed significantly from those of calf H2A and H2B. Their amino acid compositions were similar to those of the calf histones as a whole, but differed in detail in certain respects. The partial sequence of pea H2B was deduced from the amino acid compositions of BrCN cleavage fragments and tryptic peptides in comparison with the known sequence of calf H2B. It is different in the amino-terminal basic region from the calf H2B, with a blocked amino terminal and a larger number of residues. In contrast, the middle and carboxy-terminal hydrophobic regions are relatively similar, with at least 19-21 different residues and microheterogeneity at two positions of the pea sequence. The sequence of H2A may vary in much the same way as that of H2B, as suggested by the similar extent of differences in their amino acid compositions. It is thus assumed that the amino-terminal regions, at least, of H2A and H2B histones are variable in evolution provided that they remain basic enough to bind DNA, whereas the middle and carboxy-terminal hydrophobic regions of H2A, H2B, H3, and H4 should be conserved to ensure precise histone core formation inside the repeated units of chromatin.


Structural studies on the reducing-end sugar residues of polysaccharides by GLC and mass spectrometry.

The reducing-end sugar residues of pachyman, amylose, dextran, and cold water-insoluble laminaran were isolated from the polymers as penta-O-methylhexitols or penta-O-ethylhexitols by treatment with a strongly basic anion-exchange resin (OH-form) after reductjion, alkylation, and acid hydrolysis. The penta-O-alkylhexitols obtained were analyzed by GLC and combined GLC-mass spectrometry as acetates and found to be in accord with expectations based on the known structures of the polysaccharides. 3- and 4-mono-O-acetyl-penta-O-alkylhexitols, which have the same substitution pattern, were easily distinguishable on the mass spectograms, using sodium borodeuteride for reduction.


L-Lysine-alpha-ketoglutarate epsilon-aminotransferease. Properties of the bound pyridoxal 5'-phosphate.

L-Lysine-alpha-ketoglutarate epsilon-aminotransferase of Flavobacterium lutescens (Achromobacter liquidum) IFO 3084 shows positive circular dichroic bands at 340 and 415 nm where absorption maxima are observed, and fluorescence maxima at 380 and 490 nm on excitation at 340 and 415 nm, respectively. The pyridoxal 5'-phosphate absorbing at 415 nm is bound through an aldimine linkage to an epsilon-amino group of the lysine residue of the protein. Upon aging, the 415 nm pyridoxal 5'-phosphate changes to a less active form (lambda max, 325 nm), which is distinguishable from the 340 nm pyridoxal 5'-phosphate. This 325 nm bound pyridoxal 5'-phosphate is reduced with sodium borohydride and shows no circular dichroism. When the semiapoenzyme is aged under the same conditions, no spectral change is observed. These findings suggest that the pyridoxal 5'-phosphate bound through an aldimine linkage may be converted into a carbinol amine or some other related form by aging.


The carbohydrate structure of a glycopeptide released by the action of plasma kallikrein on bovine plasma high-molecular-weight kininogen.

Fragment 1, released from bovine plasma high-molecular-weight kininogen by the action of plasma kallikrein, is a glycopeptide with approximately 3 mol of each of N-acetylgalactosamine, galactose and sialic acid in one molecule. All these sugars were in the T-1 fragment obtained by tryptic digestion of fragment 1. Sialic acid can be completely released from the T-1 fragment by sialidase digestion. When this sialic acid-free T-1 fragment was incubated with purified diplococcal endo-alpha-N-acetylgalactosaminidase, all remaining sugars were released as a disaccharide. By Smith degradation and beta-galactosidase digestion, the structure of this disaccharide was found to be Gal beta 1 leads to 3GalNAc. Methylation analysis of the trisaccharide released from fragment T-1 by alkaline borohydride treatment indicated that all the galactose was obtained as the 2,4,6-tri-O-methyl derivative. Based on this evidence, the complete structure of the carbohydrate moieties of fragment 1 was proposed as Sialyl alpha 2 leads to 3Gal beta 1 leads to 3GalNAc. In addition, small amounts of a tetrasaccharide, Sialyl alpha 2 leads to 3Gal beta 1 leads to 3(Sialyl alpha 2 leads to 6)GalNAc also occurred as a carbohydrate chain of fragment 1.


Studies of peroxisomes. VIII. Evidence for framework protein of the cores of rat liver peroxisomes.

The cores of peroxisomes were purified 670 fold from a rat liver homogenate and the protein in the preparation was examined by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Two bands of protein were detected on 10% polyacrylamide gel, and their molecular weights were calculated to be about 32,000 and 27,000. On treatment of the core fraction with alkali, urate oxidase was solubilized and on 10% polyacrylamide gel this fraction gave a single band of protein with an estimated molecular weight of 32,000. These results suggests that the protein component having a molecular weight of 27,000 is the framework protein of the core of rat liver peroxisomes.


Studies on papain catalysis with substrates containing the trans-p-phenylazobenzoyl group as a probe for hydrophobic interaction.

The susceptible bonds of substrates of the type PABz-GLYn-Arg-R (R=OCH3, n=0,1,2; R=OH, n=1,2) to papain [EC 3.4.22.2] have been identified. PABz-Arg-OMe was found to  be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a Km(app) value 25 times smaller. The finding that PABz-Gly2-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S4 subsite of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S2 subsite interacts preferentially with a hydrophobic P2 residue. A negative charge on the substrate at the P2' or P3' position greatly decrease the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly2-Arg-OMe was little affected by the acridine dye proflavine.


Isolation of nonhistone-protein-rich chromatin fragments differing in composition and recovery between transcriptionally active and inactive sources.

Soluble chromatin was fragmented by deoxyribonuclease I[EC 3.1.4.5] or II[EC 3.1.4.6] digestion or sonication, followed by 0.7 m NaCl treatment, which preferentially removed DNA fragments and histones from the fragmented chromatin, but not from intact chromatin, leaving an insoluble fraction of DNA-histone-nonhistone protein complex  without appreciable redistribution of the chromatin components. Fragmentation of DNA and concentration of nonhistone proteins in the fraction were most effective with deoxyribonuclease I under the conditions used. Thus, only 1% of the DNA in transcriptionally inactive chicken erythrocyte chromatin was recovered, together with 2% of the total histone and 50-100% of the total nonhistone protein. Separate experiments showed that this DNA fraction was among the double-stranded segments having in average size of 210 base pairs and was associated with histone components responsible for the nucleosome conformation as well as most of the nonhistone protein components in the original chromatin. From transcriptionally active  chicken liver or calf thymus chromatin, however, 5-10% of the total DNA, 20-40% of the histone, and 80-100% of the nonhistone protein were recovered  in  the corresponding fraction. These results may reflect the native states and template activities in situ of various chromatins.


Fluorometric studies on the light chains of skeletal muscle myosin. I. Effects of temperature, ionic strength, divalent metal ions, and nucleotides.

Light chains of skeletal muscle myosin were studied through the reactivity of their SH groups with a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM). The experiments were carried out by reacting the reagent with myosin for a short time and measuring the amounts of reacted dye by fluorometry after separating light chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two classes of light chains, alkali light chains and DTNB light chain, were clearly distinguished by their manner of reactivity change, and differences in their environment and in their function were suggested. Although we found that the SH groups of the DTNB light chain were susceptible to very low concentrations of Mg ions (of the order of 10-5 M), we could not observe Ca2+-induced conformational change by our technique. We also estimated the stoichiometry of light chains in skeletal muscle myosin to be 1.37 mol alkali light chain 1, 1.95 mol of DTNB light chain and 0.77 mol of alkali light chain 2 per mole of myosin from the total amounts of our reagent that reacted with each light chain.


Large-scale preparation of cytosolic and mitochondrial asparatate aminotransferases from pig heart.

A procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heat muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2 g of the cytosolic isoenzyme and 1.7 g of the mitochondrial isoenzyme were obtained from 20 kg of minced heart muscle.


Studies on the synthesis of proteinase inhibitors. I. Synthesis and activity of nonapeptide fragments of soybean Bowman-Birk inhibitor.

Two heterodetic cyclic nonapeptides, X-Cys-Thr-Lys-Ser-Asn-Pro-Pro-Gln-Cys-Y (Ia: X = Ac,  Y = NH2; Ib: X = H, Y = OH), which correspond to residues 14-22 in the sequence of Bowman-Birk inhibitor, have been synthesized by Merrifield's solid-phase method. Inhibitory activities of Ia and Ib on tryptic hydrolysis of amide and ester substrates were examined. When Gly2-Lys-Gly3 and Tos-Arg-OMe were used as substrates, the values of I50 for the peptide Ia were calculated to  be 3.6 micron and 40 micron, respectively. When Gly2-Lys-Gly3 was used as a substrate, the value of Ki was calculated to be 1.5 micron. Ia was hydrolyzed slowly by trypsin, losing the inhibitory activity. When the Lys-Ser bond of Ia  was cleved with trypsin, the modified Ia could not be regenerated by trypsin. The linear peptide S, S'-dicarboxamidomethyl-Ia also was inactive and appeared to be a good substrate. Optical rotatory dispersion studies showed that the active fragments have characteristic  conformations  which  were lost upon modification to inactive derivatives.


Protein in the food prolongs DNA synthesis in the liver of rats after starvation.

After a  period on protein-free diet and then starvation,  high protein diet induces DNA synthesis in rat liver for a longer period than diet containing an equivalent amount of amino acids. The requirement for high protein diet is limited to an early part of the prereplicative period and administration of the high protein diet later did not prolong DNA synthesis.


Purification to homogeneity of spinach nitrite reductase by ferredoxin-sepharose affinity chromatography.

Assimilatory nitrite reductase was purified 1,700-fold with a yield of 22% from spinach leaves with a procedure involving ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex chromatography, gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous as shown by disc and SDS-gel electrophoresis with a specific activity (mumol NO2-reduced/min/mg protein) of 140.


Colicin E3 induced cleavage of 16s rRNA of isolated 30S ribosomal subunits.

Contrary to  the previous findings we found that a specific cleavage of 16S rRNA occurred when protein A, the active component of colicin E3, was interacted with isolated 30S ribosomal subunits at high concentrations. Necessary experiments to disprove the possibility of artefacts are described.


Metabolism of thromboxane B2 in man. Identification of the major urinary metabolite.

One and five-tenths milligrams of [3H8]thromboxane B2 (12.2 Ci/mol) was infused at a maximum rate of 6.4 microgram/min into a healthy male volunteer. Seventy-four per cent of the infused radioactivity was recovered in the urine within 13 h. Urinary metabolites of thromboxane B2 were isolated by reversed phase partition chromatography and high performance liquid chromatography. The major urinary metabolite was found to represent approximately 16.8% of the total radioactivity in the urine. The structure of this metabolite was shown by gas chromatography-mass spectrometry to be 2,3-dinor-thromboxane B2, the product of a single step of beta-oxidation. The rate of excretion of infused radioactivity and the relative percentage represented by 2,3-dinor-thromboxane B2 in the urine of man are very similar and the major urinary metabolite identical to that previously found by us in the non-human primate.


A role for aminoacyl-tRNA synthetases in the regulation of amino acid transport in mammalian cell lines.

The regulation of neutral amino acid transport was examined utilizing two temperature-sensitive aminoacyl-tRNA synthetase mutants, by comparison of their transport activities at permissive and marginally permissive temperatures. When the temperature-sensitive leucyl-tRNA -synthetase mutant tsH1 was shifted from a normal growth temperature of 34 degrees to a marginally permissive temperature for growth of 38%, a significant enhancement in the initial rate of uptake of leucine and other L system amino acids was observed when compared with that of the parental cells (CHO-S). In contrast, the uptake of A system-specific amino acids (alanine, glycine, alpha-aminoisobutyric acid) showed no significant differences relative to the parent strain. In a similar manner, a temperature-sensitive asparaginyl-tRNA synthetase mutant RJK-4 exhibited a significant enhancement of the transport activity of A system amino acids relative to its parental strain RJK-0 when the growth temperature was shifted from 33 degrees to 39.5 degrees. Preliminary kinetic data suggest that the Vmax for transport is increased when the mutants are grown under conditions of amino acid limitation. These experiments suggest that the aminoacyl-tRNA synthetase or a related product may function in the regulation of neutral amino acid transport in Chinese hamster cells.