Pulmonary vasomotion and the distribution of vascular resistance in a dog lung lobe.

We examined the influence of stellate ganglion stimulation, hypoxia, and the infusion of norepinephrine, PGF2alpha, serotonin, and histamine on the longitudinal distribution of vascular resistance and intravascular pressures in an isolated left lower lobe of the dog lung using the low-viscosity bolus technique. Sympathetic stimulation, norepinephrine, serotonin, PGF2alpha, and hypoxia increased total pulmonary vascular resistance by increasing the resistance, primarily on the arterial or upstream side of the volume midpoint, whereas histamine increased the resistance near the venous end of the lobar vascular bed. Hypoxia increased the volume upstream from the site of maximum resistance, suggesting that the larger lobar arteries were distended by the elevated lobar artery pressure. Sympathetic stimulation, norepinephrine, PGF2alpha, and serotonin, on the other hand, had little effect on the volume upstream from the maximum resistance, suggesting that these vasomotor stimuli prevented distension of the larger arteries.


Role of shell diffusion area in incubating eggs at simulated high altitude.

Embryonic development is inhibited when eggs are incubated at 9,100 m (0.3 atm) despite a normoxic environment. The problem apparently relates to respiratory gas exchange occurring by diffusion through gas-filled pores in the shell. Gaseous flux is therefore inversely proportional to ambient pressure and is affected by the physical characteristics of the ambient gas (Chapman-Enskog equation). Excess loss of H2O and CO2 occurs in eggs incubating at altitude and could be detrimental. Such increased loss should be correctable by decreasing diffusion area. This was tested by progressively increasing coverage of the shell with paraffin and incubating at simulated 0.3 ATA (225 Torr) in 100% O2. Uncoated eggs failed to hatch, but numbers of chicks increased with increased coverage. Maximum hatch was an extrapolated 90% of controls at 69% shell coverage. With further coverage, hatch size decreased. Egg weight loss, and estimate of H2O diffusion, was around three times controls in uncoated eggs but decreased linearly with paraffin coverage, reaching near normal at maximum hatch. Reduction of diffusion area to 0.3 normal at maximum hatch generally balanced the increased flux predicted for 0.3 ATA.


Exercise ventilation correlates positively with ventilatory chemoresponsiveness.

To determine the relationship of ventilatory responsiveness to hypoxia and hypercapnia to exercise hyperpnea, these responses and steady-state exercise ventilation (VE) were measured in 16 athletes during light (1/3 VO2 max) and heavy (2/3 VO2 max) exercise. Both the hypoxic and hypercapnic ventilatory responses correlated positively with VE per unit metabolic rate (VE/VCO2) at both exercise levels (P less than 0.05). The contribution of the hypoxic response to normoxic exercise VE was quantified by comparing VE in normoxia to VE during a brief (1 min) exposure to high O2 (PAO2 = 200 Torr). High O2 reduced normoxic exercise VE by a mean of 20% at either exercise intensity. Among individuals this reduction was directly dependent upon the intensity of the hypoxic response, and ranged from 7 to 42% of normoxic VE. After the variable reduction of normoxic VE by hyperoxia, all correlations of ventilatory response with exercise VE were lost except for the correlation of hypercapnic response with heavy exercise VE/VCO2. These findings indicate that the extent of VE in light or heavy exercise is modified by the strength of the hypoxic ventilatory response, and that the hypercapnic response independently correlates with VE during heavy exercise.


Role of cardiac output in the pressor responses to graded muscle ischemia in man.

Ten men repeatedly performed leg exercise (100-150 W) for 7 min with 30-min recovery periods interspersed. Both legs were made ischemic by total occlusion (OCCL), first for 3 min immediately after exercise and second for 30 s before exercise ended and 3 min into recovery. In addition legs were occluded for 3 min at rest (seated). OCCL at rest increased mean arterial pressure (MAP) by 9 Torr but did not affect cardiac output (CO) or heart rate (HR). OCCL at the end of exercise significantly raised MAP and HR above control values during 3-min recovery but CO was unaffected. OCCL 30 s before the end of exercise further increased MAP and HR significantly during recovery; MAP, CO, and HR were significantly increased above control values (CO by 2.1 1-min-1) during the 3rd min of recovery. We conclude that a strong reflex from ischemic legs maintains normal or elevated CO during leg OCCL. Thus CO was too high relative to total vascular conductance so that MAP was elevated.


Effects of body position change on thoracoabdominal motion.

With a linearized respiratory magnetometer, measurements of anteroposterior and lateral diameters of both the rib cage and the abdomen were made at functional residual capacity and continuously during tidal breathing. Twenty-five subjects with normal respiratory systems were studied in the sitting, supine, lateral decubitus, and prone body positions. When subjects changed from sitting to supine position anteroposterior diameters of both rib cage and abdomen decreased while their lateral diameters increased. Both anteroposterior and lateral tidal excursions of the rib cage decreased; those of the abdomen increased. When subjects turned from supine to lateral decubitus position both anteroposterior diameters increased and the lateral diameters decreased. This was associated with an increase in both lateral excursions and a decrease in the abdominal anteroposterior excursions. Diameters and tidal excursions in the prone position resembled those in the supine position. Diameter changes could be explained by gravitational effects. Differences in tidal excursions accompanying body position change were probably related to 1) differences in the distribution of respiratory muscle force, 2) differences in the activity or mechanical advantage of various inspiratory muscles, and 3) local compliance changes in parts of the rib cage and abdomen.


Mechanisms of pulsus paradoxus in upper airway obstruction.

Pulsus paradoxus, a greater than 10 Torr systolic pressure fluctuation during the respiratory cycle, is seen in upper airway obstruction. To test the hypotheses 1) that blood is pooled in the pulmonary circulation with reduced return to the left heart during inspiration and 2) that inspiration increases left ventricular afterload, the following was done. Esophageal pressure (Pes), pericardial pressure (Pp), left atrial transmural pressure (Platm), and left ventricular transmural pressure at end-isovolumic systole (Plvtm) were recorded during partially obstructed inspirations in spontaneously breathing dogs anesthetized with pentobarbital (25 mg/kg). Changes in Pes and Pp were nearly identical (r = 0.9883) confirming that changes in Pes adequately reflect changes in Pp. During inspiration Platm and Plvtm increased 0.5 and 0.4 Torr, respectively, per Torr decrease in Pes suggesting that increased blood return to left atrium and increased left ventricular afterload occur. Similar changes were observed during near constant thoracic volume (total airway block) and cardiac reflex blockade (atropine 0.05 mg/kg and propranolol 0.5 mg/kg). Thus mechanical factors including left ventricular afterload appear of major importance in producing pulsus paradoxus in upper airway obstruction.


Effects of exercise training on coronary reactive hyperemia and blood flow in the dog.

The reactive hyperemic responses to 10-s coronary occlusions were studied in seven sedentary-control (C) and eight exercise-trained (T) anesthetized dogs, with electromagnetic flowmeters placed on the left anterior descending coronary artery. Radiolabeled microspheres (9 +/- 0.8 micron) were used to measure resting coronary flow per gram and to study the effects of isoproterenol infusion (ISO) (1 mg/kg-min) on total and regional coronary flow. Base-line coronary flow per 100 g was significantly greater in the T dogs (122 +/- 7) than in C dogs (100 +/- 4). During ISO, T and C coronary resistances did not differ significantly, whereas the effects of ISO on total and regional coronary flow were quite different in T dogs as compared to C dogs. C and T hyperemic flow debt repayments did not differ significantly; however, the peak reactive hyperemic flow in T dogs (344 +/- 12%) was significantly greater than the control (306 +/- 10%). Since resting coronary flow per gram was greater in T dogs, the greater peak reactive hyperemic flow implies that T dogs have an increased coronary reserve. Although the mechanisms involved are unclear, the results of this study indicate that chronic exercise training may have beneficial effects on coronary physiology.


Relaxed +GZ tolerance in healthy men: effect of age.

Fifty-three healthy US Air Force aircrewmen, 26-55 yr old, volunteered for a centrifuge study designed to determine the effect of age on relaxed +GZ tolerance. Each was subjected to G forces of gradual and rapid onset, with G tolerance determined by standardized contraction of peripheral visual fields. Of the subject characteristics studied, only age was positively correlated with rapid-onset G tolerance; both age and weight were positively correlated with gradual-onset G tolerance. A combination of age and weight gave a stronger positive correlation with G tolerance (rapid- and gradual-onset) than did either characteristic alone. No significant negative correlations were observed. We conclude that aging may offer some protection from G stress; there is no evidence that aging leads to a decrement in G tolerance.


A respiratory jacket for ventilatory measurements in children.

The properties of an air-filled polyurethane-coated nylon jacket for measuring volume parameters were determined on a model and in 13 subjects. There was a linear relationship between jacket pressure on a logarithmic scale and the lung volume change over an operating pressure range of 0.7-6 cmH2O. The magnitude of pressure change was frequency dependent. The magnitude of a calibration signal was reduced by superimposed tidal breathing. For the jacket tested, the calibration signal during tidal breathing was 87% of the calibration during apnea. With these properties established, a calibration technique was devised and tested on 13 infants. Corrected tidal volumes (VT) obtained with the jacket correlated well with those obtained with a pneumotachograph (r = 0.986). At the operating pressures used the jacket caused no significant change in functional residual capacity (FRC), VT, or frequency. The jacket is a reliable technique for measurement of VT and immediate changes in FRC in awake children.


An electrode for PN2O and PO2 analysis in blood and gas.

The development of a new polarographic sensor for measuring simultaneously both N2O and O2, in either gas or blood, is described. The cathode is made of silver, and it is shown that silver deposition on normal platinum or gold cathode electrodes can result in an enhancement of a PO2 signal, when measured in the presence of nitrous oxide. Silver can be deposited on the cathode by means of Ag+ ions diffusing through the electrolyte from an Ag/AgCl reference electrode. The use of an Ag cathode enables both O2 and N2O signals to be measured.


A simple technique for measuring alveolar CO2 in infants.

We designed a system consisting of a nostril adapter and a catheter for measurement of end-tidal ("alveolar") CO2 in small infants using the Beckman LB-1 analyzer. Using this system, we obtained a capillary-"alveolar" PCO2 difference (PaCO2-PACO2) of 2.4 Torr in 19 normal term infants, 3.5 Torr in 12 preterm infants who had recovered from respiratory distress syndrome, and 9.0 Torr in 4 preterm infants with bronchopulmonary dysplasia. We also found in 5 infants that systems using faster flow rates (140-400 ml-min-1) or slower response times (greater than 0.3 s) reduced the duration of the alveolar plateaus as well as the calculated end-tidal PCO2. In addition, when term infants were not in a steady state, PACO2 values were unreliable and significantly lower than capillary values. We therefore conclude that measurement of alveolar CO2 using our technique is a simple noninvasive way of evaluating newborn pulmonary function.


Effect of interval between booster vaccination and time of breeding on protection against campylobacteriosis (vibriosis) in cattle.

Thirteen heifers (group A) given a booster vaccination with Campylobacter fetus 4 1/2 months before breeding to bulls infected with the homologous strain had a pregnancy rate of 54%, compared with a pregnancy rate of 92% for 12 heifers (group B) given a booster vaccination 10 days before the 63-day breeding period began. None of 7 nonvaccinated heifers became pregnant, and all remained infected for at least 136 days. All three groups of heifers were bred by the same infected bulls. Nine of the heifers in group A, but none of the heifers in group B, became infected during the first estrus. Blood serum agglutination titers peaked 2 weeks after the first vaccination but  decreased to prevaccination titers 6 weeks later. Titers more than double those produced by one vaccination peaked between 2 and 3 weeks after booster vaccination but in most heifers had decreased to low titers 7 weeks later. When the breeding period began, the median antibody titer was 640 for heifers in group A and 10, 240 for heifers in group B. Indications of an anamnestic response due to natural challenge were not found in any of the 9 infected heifers in group A when serum titers were determined 27 and 34 days after breeding began. Therefore, it appears that in many heifers and cows, only the antibodies produced by booster vaccination will be available to provide protection. Inasmuch as vaccinal antibody titers decrease rapidly, it was concluded that booster vaccinations should be given approximately 10 days before breeding so that titers will be high during the breeding period.


Evaluation of a transport medium for campylobacter (Vibrio) fetus.

A quantitative assessment was performed on a serum enrichment medium for Campylobacter (Vibrio) fetus, to test its effectiveness as a transport medium. Abundant yields of C fetus were obtained with an inoculum size of 10(4), even after keeping the medium in air at 22 to 23 C for 4 days prior to incubation at 37 C under microaerophilic conditions. The organism could be isolated from the medium kept in air at 22 to 23 C for at least 2 days, with an inoculum as small as 100 organisms. It was concluded that this formulation comprises a convenient and effective transport medium for field use. Transit time should not exceed 3 days and, ideally, should be restricted to less than or equal to 2 days. At least duplicate vials of transport medium should be inoculated with each sample.


Anomalous left coronary artery in a calf.

An anomalous left coronary artery was seen arising from an ostium in the pulmonary artery in a 4-month-old Hereford calf. Endocardial fibrosis was found in the left atrium and left ventricle. The mitral valve was dilated and thickened. A normal right coronary artery originated from the aorta.


Age and milk production data of cattle culled from a dairy herd with paratuberculosis.

Statistical assessment of age and milk production data revealed a significantly shorter life expectancy and reduced milk production of Mycobacterium paratuberculosis-infected dairy cows, when compared with non-infected herdmates. High producing cows were frequently culled after their 1st or 2nd gestation, contributing to an undetermined economic loss with regard to their potential breeding value. Cows with subclinical infection frequently had problems of infertility and of mastitis.


Azotemia in cattle.

Blood biochemical studies were performed on 21 azotemic cattle with various disease conditions. The cattle were greater than or equal to 4 months old. Blood urea nitrogen concentrations ranged from 41 to 346 mg/dl. The azotemia was determined to be of prerenal origin in 10 cattle and of renal or postrenal origin in 11 cattle. The most consistent biochemical finding was that of hypochloremia. Sixteen cattle had lower than normal concentrations of serum calcium, and 15 had serum inorganic phosphorus concentrations greater than normal. In 17 cattle, the serum concentration of inorganic phosphorus was greater than that of calcium. Plasma sodium content was lower than normal in 9 of the 21 cattle, and plasma potassium concentrations were quite variable. Most of the cattle tended to maintain a normal acid-base status or had metabolic alkalosis. It was concluded that evaluation of biochemical data does not make it possible to determine whether azotemia is prerenal, renal, or postrenal in origin.


Induced paragonimiasis in cats: clinical signs and diagnosis.

Twenty-eight specific-pathogen-free cats were inoculated with 14 to 50 metacercariae of Paragonimus kellicotti obtained from the hearts of naturally infected crayfish. Young flukes excysted in the intestine of cats and appeared in the peritoneal cavity from 1 to 14 days after inoculation (DAI) and in the pleural cavity from 5 to 23 DAI. Flukes penetrated the pulmonary parenchyma and formed hemorrhagic subpleural lesions within 5 weeks after inoculation. Marked eosinophilia developed between 2 and 12 weeks after inoculation. Fluke-containing pulmonary lesions were detected by radiography 3 to 4 weeks after inoculation. Lesions developed most frequently in the right caudal lung lobe. Clinical signs were mild and did not appear until 4 weeks after inoculation. Thereafter, cats appeared dull and coughed intermittently. One cat became dyspneic due to pneumothorax. Paragonimus eggs were first detected at the 34th DAI, using a fecal sedimentation technique.


Portal hypertension secondary to a right atrial tumor in a dog.

A German Shepherd Dog was examined because of ascites and hepatomegaly. The finding of proteinaceous ascitic fluid and hepatic histologic features of centrizonal sinusoidal congestion suggested hepatic venous outflow obstruction. Diagnostic tests eliminated heart failure from the differential diagnosis and thus indicated an obstruction between the right atrium and hepatic sinusoids. Vascular manometry and angiography established the site of the lesion in the right atrium and confirmed portal vein hypertension. Surgical exploration revealed an unresectable right atrial tumor occluding the caudal vena cava.


Acquired achalasia (megaesophagus) in a dog: clinical features and response to therapy.

Acquired megaesophagus was diagnosed in a mature mixed-breed dog. The onset of clinical signs, pattern of esophageal motility, and response to surgery differed from these factors in dogs with congenital megaesophagus. The dog improved following esophagomyotomy and feeding from an elevated container. Emptying of the esophagus improved postsurgically.


Lymphangioma in four dogs.

Lymphangioma, a rare benign tumor of lymph vessels, was diagnosed in 4 dogs. The lesions were in the nasopharynx, retroperitoneum, axilla, and medial aspect of thigh. Treatment was either surgical excision or marsupialization.


Surgical approach for medial meniscectomy in the horse.

After preliminary studies on cadavers, a surgical approach was developed for removal of the medial meniscus, offering maximal exposure and minimal trauma to the articular surfaces of the medial tibial plateau and femoral condyle. Medial meniscectomy was then performed on 10 clinically normal horses that had been given general anesthesia. Nine horses were euthanatized at intervals of 1 to 6 months, and the joints were evaluated grossly and histologically. (One horse will be examined 1 year after surgery). Medial meniscus tears were diagnosed in 5 horses; medial meniscectomy was done on 4 horses and 1 horse was rested. Removal of the medial meniscus was considered feasible in the horse, but the resulting degree of locomotor impairment varies. Prognosis for surgical removal of the medial meniscus was considered to be guarded to good.


Clinical and clinicopathologic observations in induced malignant catarrhal fever of cattle.

In an epizootic of malignant catarrhal fever in 1976/1977 in southern California, 166 cows (17%) died. Blood from one of those cows was used in transmission studies involving 8- to 14-month-old steers. In 17 (74%) of 23 inoculated steers, the disease was reproduced. The clinical signs in 8 of the diseased steers were characterized by an initially mild intestinal form of the disease, which suddenly fulminated and terminated in death within 1 to 3 days. No lesions or only mild oral or ocular lesions were noticed in this group, but in the other steers, such lesions predominated. It was concluded that a subtle form of the disease can terminate in acute fatal indigestion.


Effect of local anesthesia of the penis and dorsal penile neurectomy on the mating ability of bulls.

Eleven bulls were used to study the effect of the loss of nerve sensitivity in the free portion of the penis and glans penis on their ability to copulate. The loss of sensitivity was induced by topical anesthesia, infiltration anesthesia, and dorsal penile neurectomy. Topical anesthesia of the glans penis reduced the ability of the bulls to search for and to locate the vagina and to complete copulation; however, all bulls eventually copulated. Topical anesthesia of the free portion of the penis and glans penis blocked the ability of all bulls to copulate. Infiltration of the glans penis with lidocaine resulted in bulls failing to ejaculate, indicating the need for input from deep nerve receptors. Ten of the eleven bulls with unilateral neurectomy were able to copulate, but a longer time was required. In the one bull that failed to complete intromission and ejaculation, the sectioned nerve made up 75% of the nerve mass to the distal end of the penis, leaving only a small number of functional fibers. With this small number of fibers, there may have been insufficient sensory input to elicit the thrusting responses and to cause the animal to ejaculate. Bulls with bilateral neurectomy were unable to copulate.


Evaluation of phosmet for the control of the common scabies mite on cattle.

Thirty-four young range cattle heavily infested with the common scabies mite, Psoroptes ovis, were dipped in phosmet (O,O-dimethyl phosphorodithioate S-ester with N-(mercaptomethyl)phthalimide) in 15 trials. All concentrations from 0.15% to 0.25% that were applied once failed in at least one trial, but all concentrations from 0.075% to 0.20% were successful in eradicating mites when used twice at 7- to 10-day intervals. One single dip tried at 0.30% also was successful. Uninfested yearlings were dipped in 0.30% phosmet without apparent intoxication, but 2-year-old cattle treated in a spray-dip machine at 0.40% active ingredient became depressed and stiff gaited.


Acquired bilateral cortical cataracts in mature sows.

Bilateral cortical cataracts developed in approximately 30% of all sows greater than or equal to 2 1/2 years old on 4 farms in southwestern Ontario and on 2 farms in Quebec. A cataractogenic agent was not established, but hygromycin B was suspected as the cause.


Repair of fractured dens of the axis in a foal.

Quadriplegia of 4 days' duration in a 3-week-old foal was caused by displacement of the dens of the axis due to a fracture at the dens growth plate. The displacement was reduced and was immobilized surgically. Two months after surgery, the foal was able to rise, walk, and trot without apparent neurologic deficiencies. Three years later, the only defect was a mild gait disturbance in the left hindlimb seen at a canter and a gallop.


A new route to the synthesis of cyclitol derivatives.

Three cyclitol derivatives, 2,4/3-triacetoxycyclohexanone diethyl dithioketal (XVIII), and 2,3,4-tri-O-acetyl-1-O-ethyl-2,4/1,3-cyclohexanetetrol (XXA) and 2,3,4-tri-O-acetyl-1-O-ethyl-1,2,4/3-cyclohexanetetrol (XXB), have been synthesized via the cyclisation of 2,3,4-tri-O-acetyl-5,6-dideoxy-D-xylo-hex-5-enose diethyl dithioacetal (XIV) and the cyclisation of 2,3,4-tri-O-acetyl-5,6-dideoxy-D-xylo-hex-5-enose diethyl acetal (XV) under ultraviolet or direct sunlight, in aqueous acetone.


Cephamycin derivatives: comparison of the in vitro and in vivo antibacterial activities of SQ 14,359, CS-1170, and cefoxitin.

SQ 14,359 is a new cephamycin-type (7alpha-OCH3) antibiotic belonging to a series containing a 7alpha-ureidoacetyl substituent. The compound is the most potent extended spectrum derivative of this type yet reported, surpassing CS-1170 and cefoxitin by a wide margin. This activity in vitro which extends throughout the Enterobacteriaceae is particularly prominent against Gram-negative organisms that are producers of "cephalosporinase-type" beta-lactamases such as Enterobacter, Serratia, Citrobacter and indole-positive Proteus species. Superior activity also is demonstrated in vitro against streptococci, beta-lactamase-producing staphylococci, Haemophilus influenzae, Neisseria gonorrhoeae, and many Gram-negative pathogens resistant to aminoglycoside antibiotics. Experimental chemotherapeutic studies have confirmed these observations in wound and selected systemic infections in mice as well as acute pyelonephritis and meningitis in rats. The pharmacokinetics for each drug including antibiotic bound to serum was similar in both mice and rats. The pharmacokinetic profile in blood and cerebrospinal fluid favored SQ 14,359.


Action of cefoxitin and cefamandole on human neutrophil function.

Polymorphonuclear neutrophilic leukocyte chemotaxis was examined in vitro in the presence to two new antibiotics: cefamandole and cefoxitin. Results indicate that cefamandole inhibited neutrophil chemotaxis to a significant degree only at high antibiotic concentrations of 100 microgram/ml (P less than 0.01) and has no significant effect at normal serum therapeutic range. Cefoxitin was found to produce a 43% inhibition (P less than 0.01) of human in vivo neutrophil chemotaxis at antibiotic concentrations of 100 microgram/ml and have a minimal inhibitory effect (1 approximately 9%) at low concentrations (1 approximately 5 microgram/ml). Both cefamandole and cefoxitin had no significant effect on opsonophagocytosis.


The chemistry of the rubradirins. I. The structures of rubransarols A and B.

The antibiotic rubradirin, C48H46N4O20 was cleaved at an ester function by aqueous methylamine into rubransarol A, C23H23NO8, and a methyl amide, C26H28N4O12. Rubradirin B, C40H33N3O15, was similarly cleaved in methanolic ammonia into rubransarol B, C23H23NO8, and the primary amide, C17H13N3O7. The rubransarols are shown to be unique ansamycins which are isomeric at a double bond in the large ring.


Trichostatin C, a glucopyranosyl hydroxamate.

In addition to trichostatins A and B, a new antifungal antibiotic, trichostatin C was isolated. The structure was shown to be a glucoside of trichostatin A by spectroscopic examinations and chemical degradations. Trichostatin C is presumably the first example of a glucopyranosyl hydroxamate from nature.


The isolation and characterization of rubradirin B.

Rubradirin B, C40H33N3O15, was separated from other components of the rubradirin complex by chromatographic and crystallization procedures. The spectrum of antibacterial activity is similar to that of rubradirin, but the antibiotic is less active.


Rifamycin R, a novel metabolite from a mutant of Nocardia mediterranea.

Rifamycin R is a novel ansamycin produced by a mutant of Nocardia mediterranea; both physical and chemical data indicate that it is 30-demethyl-30-hydroxymethyl rifamycin S.


G1499-2, a new quinoline compound isolated from the fermentation broth of Cytophaga johnsonii.

A new quinoline compound, G1499-2[C18H21NO(I)] is produced by Cytophaga johnsonii. G1499-2 has an unusual structure containing a cyclopropylidene radical. The compound has limited antibiotic activity against a few bacteria. It is not toxic to mice.


Production of deoxyfrenolicin and a new antibiotic, frenolicin B by Streptomyces roseofulvus strain AM-3867.

Two antibiotics of frenolicin group, antibiotic AM-3867 I and II were isolated from the fermentation broth of Streptomyces roseofulvus strain No. AM-3867, a soil isolate. The former was a new antibiotic designated as frenolicin B and its structure containing gamma-lactone was determined, while the latter was identified as deoxyfrenolicin having been chemically prepared from frenolicin.


Carbon-13 nuclear magnetic resonance assignments of pactamycin and related compounds.

All resonances observed in the 13C NMR spectrum of the antitumor antibiotic pactamycin and its degradation product pactamyçate have been assigned, employing off-resonance and specific proton decoupling as well as comparison with the 13C NMR spectra of the model compounds m-aminoacetophenone and ethyl 6-methylsalicylate.


Ionomycin, a new polyether antibiotic.

Ionomycin, a new polyether antibiotic with a high affinity for calcium ions, is obtained in pure form from fermentation broths of Streptomyces conglobatus sp. nov. Trejo by solvent extraction. It is unique amongst known polyether antibiotics in that it has a UV absorption maximum at 300 nm. thereby distinguishing it from other antibiotics of its class. The Ca salt has the molecular formula C41H70O9Ca. Ionomycin is a narrow spectrum antibiotic being active against Gram-positive bacteria.


Noboritomycins A and B, new polyether antibiotics.

Noboritomycins A and B, two new polycyclic ionophoric polyethers were isolated from a strain of Streptomyces noboritoensis. The crystal structure and absolute configuration of noboritomycin A were established by X-ray analysis of its silver salt C43/63O14Ag. Noboritomycin A is the first metabolic polyether possessing two carboxylic acid functions on the carbon backbone (C-31), namely a free acid and an additional carboxylic acid ethylester group. An unusual spiroketal system as well as a salicylic acid chromophore represent further remarkable elements. Noboritomycin A shows in this respect a structural relationship to salinomycin and lasalocid respectively. Comparison of physico-chemical data, in particular the interpretation of the 1H- and 13C-NMR spectra, revealed that noboritomycins A and B are structurally closely related, noboritomycin B carrying an ethyl substituent on the aromatic ring in the place of a methyl group present in noboritomycin A. Both metabolites exhibit activity against Gram-positive bacteria and against Eimeria tenella (chicken coccidiosis).


New antibiotics, enaminomycins A, B and C. I. Producing organism, fermentation and isolation.

New antibiotics, enaminomycins A, B and C, were found in the culture broth of streptomycete strain No. 13120, which was identified as Streptomyces baarnensis and designated as S. baarnensis No. 13120. Fermentation of enaminomycins were performed by conventional submerged culture in a 30-liter jar fermentor. Isolation of the antibiotics was performed by centrifugation of the culture broth and adsorption of the antibiotics from the supernatant on a column of activated carbon, followed by elution with aqueous acetone. Enaminomycins A, B and C were separated from each other on a column of Sephadex LH-20.


New antibiotics, enaminomycins A, B and C. III. The structures of enaminomycins A, B and C.

The structures of enaminomycins A and B were determined by their physico-chemical properties and X-ray crystallographic analyses to be 4-amino-2,5-dioxo-7-oxa-bicyclo[4,1,0]hept-3-ene-3-carboxylic acid and 2-oxo-4-amino-5-hydroxy-5-acetonyl-7-oxa-bicyclo[4,1,0]hept-3-ene-3-carboxylic acid, respectively. The structure of enaminomycin C was also determined by the analysis of NMR spectrum and other physico-chemical properties to be 2-oxo-4-amino-5-hydroxy-7-oxa-bicyclo[4,1,0]hept-3-ene-3-carboxylic acid.


Production, isolation and chemical characterization of mimosamycin.

A procedure is described for the large-scale production of mimosamycin, a satellite antibiotic found in the culture filtrate of Streptomyces lavendulae No. 314. The 1H and 13C NMR, and mass spectroscopic data, are explained in terms of the structure of mimosamycin.


Mechanism of action of macromomycin: DNA strand scission, inhibition of DNA synthesis and mitosis.

The effects of macromomycin (MCR), a high molecular weight peptide antibiotic, on cell division, DNA synthesis and DNA fragmentation were examined in cultured mammalian tumor cells. When MCR was added to HeLa cell culture simultaneously with [3H]thymidine, inhibition of DNA synthesis was observed depending on the amount of the drug present, although the inhibition was partial even at a high concentration of the drug. Preincubation of cells with MCR for 2 hours before assay was required for the complete inhibition of DNA synthesis. Cell division of synchronized L5178Y cells, arrested at metaphase, was strongly inhibited by MCR, indicating that the inhibition of cell mitosis by the drug was not dependent on the inhibition of DNA synthesis. Strand scission of DNA in MCR-treated cells was observed by alkaline sucrose gradient centrifugation. The fragmentation of cellular DNA occurred at low concentration of the drug and within a very short incubation time (37 degrees C, 5 minutes). At high concentrations of the drug, however, the size of the fragmented DNA remained constant. DNA polymerase activity in isolated nuclei from HeLa and L5178Y cells was stimulated by MCR. These data suggest that MCR works directly on cell nuclei and strand scission of DNA is one of the more important actions of the drug.


Studies on the ionophorous antibiotics. XVI. The ionophore-mediated calcium transport and concomitant osmotic swelling of mitochondria.

The effects of various carboxylic ionophores on divalent metal cation translocation in mitochondria have been investigated. High levels of divalent cation ionophores lysocellin and lasalocid A (10 approximately 50 micrometer) produced mitochondrial osmotic swelling in Ca2+ or Mg2+ medium, which was associated with an increase of cation influx. The extent of swelling was a function of both the ionophore and cation concentrations in the medium. This effect was larger in mitochondria de-energized by treatment with antimycin A and oligomycin than in respiring mitochondria. On the other hand, the monovalent cation ionophores carriomycin and etheromycin at concentrations of 50 approximately 100 micrometer also induced mitochondrial swelling in Ca2+ medium but were ineffective in Mg2+ medium. Addition of ruthenium red reversed divalent cation ionophore-induced swelling and released Ca2+ from preloaded mitochondria. In contrast, ruthenium red increased monovalent cation ionophore-induced swelling. In a divalent cation-free medium, lysocellin and lasalocid A caused depletion of membrane-bound Ca2+ and released endogenous Ca2+ and Mg2+ from mitochondria, while carriomycin and etheromycin exerted only a limited effect. These results indicate that the divalent cation ionophores affect divalent cation distribution in mitochondria by increasing both influx and efflux of the cations through the inner membrane.


Physiological disposition of a series of rifamycins in rat: a comparative study.

The disposition of four C3-substituted piperazinyl rifamycins was studied in the rat following the intravenous administration of 5 mg/kg of the 14C-labelled antibiotics. Considerable quantitative differences in the pharmacokinetics of these antibiotics were shown in blood levels, tissue distributions and body clearances. Feces were largely the major route of elimination for the parent drug and metabolites. The results suggest that the liver compartimentalization, regulating the biliary excretion, is to be the kinetic parameter affecting the pharmacokinetic behaviour of this class of antibiotics.


Effects of cycloheximide and streptovitacin A on protein synthesis and gastric secretion in rats.

Cycloheximide and streptovitacin A administered in vivo to rats display a similar dual effect on the labelling of soluble liver proteins by valine-14C, and result in a similar enhancement of liver uridine kinase activity. On the other hand, in pylorus-ligated rats, both antibiotics markedly depress gastric secretion, acid output, and the level of mucoproteins and proteolytic activity in secreted juice. Streptovitacin A on a molar basis was in all cases 5 approximately 8 times more effective than cycloheximide.


Heterogeneity and distribution of lipopolysaccharide in the cell wall of a gram-negative marine bacterium.

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.


Effect of light nitrogenase function and synthesis in Rhodopseudomonas capsulata.

The metabolic versatility of the purple nonsulfur photosynethetic bacterial permits the expression of either a phototrophic or a dark aerobic mode of growth. These organism also possess nitrogenase activity which may function under semiaerboic conditions. On the basis of these important properties, the light dependence of nitrogenase function and synthesis in Rhodopseudomonas capsulata was investigated. Nitrogenase activity was strictly dependent on light; no activity was observed in the dark, even when energy (ATP) was supplied by oxidative phosphorylation. It was concluded that the low-potential reducing agent required by the nitrogenase-catalyzed reaction could only be generated by a photochemical reaction. Nitrogenase biosynthesis was also largely dependent on light; however, a small amount of synthesis was observed in resting cells incubated in the dark. Resting cells prepared from dark-grown cultures synthesized nitrogenase at high rates upon illumination. The highest stability of nitrogenase in these resting cells was observed when suspensions were exposed to a diurnal pattern of illumination rather than continuous light. Although nitrogenase function and synthesis are closely coupled to photosynthetic activity, the biosyntheses of bacteriochorophyll and nitrogenase are independent of each other and are most probably subject to different regulatory mechanisms by light.


Modulation of an apparent mRNA pool for extracellular protease in Bacillus amyloliquefaciens.

Late-log-phase cells of Bacillus amyloliquefaciens have the unusual capacity to produce extracellular protease for over 60 min in the presence of rifampin or actinomycin D at levels which strongly inhibit incorporation of amino acids into cellular protein. If cells are incubated in the presence of high levels of amino acids for 75 min this capacity is exhausted, but it is retained if the incubation is carried out in low levels of amino acids. Transfer of exhausted cells from high to low concentrations of amino acids results in a progressive recovery of the capacity for rifampin-actinomycin-insensitive protease production. The results seem best explained on the basis of the accumulation of a reserve pool of mRNA for extracellular protease. Measurement of the apparent mRNA pool size over 12 h shows a cyclical rise and fall, and these changes correlate with a periodic variation of the rate of protease production. A working hypothesis is presented to account for these observations in terms of a novel control situation over protease mRNA transcription.


Crossed immunoelectrophoresis, in the presence of tween 20 or sodium deoxycholate, of purified membrane proteins from Acholeplasma laidlawii.

Five membrane proteins from Acholeplasma laidlawii have been previously purified on a large scale. These proteins have been used to establish the relationship between the precipitation lines obtained by crossed immunoelectrophoresis of solubilized cell membrane proteins from A. laidlawii in the presence of the neutral detergent Tween 20 or those obtained in the presence of the anionic detergent sodium deoxycholate. This relationship, which was unambiguously established for four of the five proteins, was determined by tandem or "parallel" crossed immunoelectrophoresis of the sodium deoxycholate-solubilized membrane together with the purified proteins. Membranes from strain A of A. laidlawii were composed of proteins, which were immunologically related to and probably identical to membrane proteins from strain B of this organism.


Kinetics of accumulation of a photodynamically induced cell-surface polypeptide in a species of Arthrobacter.

Cells of a species of Arthrobacter were incubated in the light with methylene blue, a dye that sensitizes photooxidative reactions by the production of singlet oxygen. An early and major response by the cells to these conditions was stimulation of synthesis of a single cell-surface polypeptide, 21,000 daltons in mass. The rate of synthesis of this polypeptide reached a maximal level about 30 min after the start of illumination. As a consequence, the amount of this polypeptide increased at least 10-fold during a period of 5 h. The presence of histidine or methionine, scavengers of singlet oxygen, markedly diminished synthesis and accumulation of this polypeptide. Concomitant with the accumulation of this polypeptide on the cell surface was the appearance of an extensive array of pili.


Characterization and construction of molecular cloning vehicles within Staphylococcus aureus.

Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.


Lack of lysogenic induction in "diaminopimelic acid spheroplasts".

As part of an attempt to develop a semi-in vitro system of lysogenic induction, using spheroplasts of Escherichia coli K-12 lysogenic for prophage lambda, we prepared spheroplasts by depriving E. coli dap of diaminopimelic acid (DAP-spheroplasts). DAP-spheroplasts made from E. coli (lambda cI857) were thermally inducible. However, DAP-spheroplasts of E. coli (lambda) were not inducible by UV light. Thus, it appears that a functional cell wall is required for UV induction of prophage lambda.


Linkage analysis of Pseudomonas glycinea.

The IncP-1 plasmid R68 and variants R68.45 and R68.185 were tested for their chromosome donor ability in a selected recipient of Pseudomonas glycinea PGR12. It was found that variants did not express their selected characteristic of increased donor ability over that of R68 or R68.5, our commonly used donor plasmids. Coinheritance analysis of a variety of crosses provides evidence of a linkage group comprising 11 loci.


Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation.


Effects of magnesium, calcium, and serum on reversion of stable L-forms.

The L-form of Agromyces ramosus was stable in the absence of penicillin when transferred on heart infusion agar containing NaCl and serum. It reverted to its bacterial form, however, when magnesium replaced the serum in this medium. On a dilute medium containing NaCl but lacking serum, the L-form died out unless calcium, magnesium, or serum was added. It grew as the L-form in the presence of calcium of serum but reverted to the bacterial form in the presence of magnesium. Reversion also occurred when magnesium was added to the dilute medium containing serum. Calcium interfered with or prevented the magnesium-induced reversion. The revertant bacterial form resulting from these studies was not NaCl sensitive, as was the case of the bacterial revertant of this organism produced in soil (A. H. Horwitz and L. E. Casida, Jr., Can. J. Microbiol, 24:50--55, 1978).


Carbon monoxide oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum.

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO(2). Rates up to 0.4 mumol min(-1) mg of wet cells(-1) were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO(2) reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 mumol min(-1) mg of protein(-1) (35 degrees C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was "ping-pong," indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO(2) to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO(2) do not possess a CO-oxidizing system.


Role of metalloprotease in activation of the precursor of staphylococcal protease.

A metalloprotease was isolated from the culture medium of a mutant of Staphylococcus aureus strain V8. The enzyme had a molecular weight of 38,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an optimum pH of 7.0 and exhibited a specificity for peptide bonds on the N-terminal side of large hydrophobic residues. The protease was fully inactivated by 0-phenanthroline but could be reactivated by zinc ions. Cobalt may be substituted for zinc, producing an activity which corresponds to 160% of that of the native enzyme. All these data indicate that this protease is a typical bacterial neutral metalloprotease. The role of this metalloprotease in the activation of the precursor of another protease secreted by the same organism, staphylococcal protease, has been identified. Mutants which lack the metalloprotease accumulated the precursor, which can be specifically activated by the addition of the purified metalloprotease or the related enzyme thermolysin. The purification of the precursor is also reported.


Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.


Coordinated regulation of octopine degradation and conjugative transfer of Ti plasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons.

By using the analog noroctopine, mutants of agrobacterium tumefaciens were isolated with altered regulation patterns for the Ti plasmid-borne octopine utilization genes. These could be divided into three classes: (i) strains with a constitutive level of octopine enzymes and a high degree of spontaneous Ti transfer; (ii) one strain with constitutive octopine enzymes but no spontaneous Ti transfer; and (iii) strains with an altered inducibility in which, contrary to the wild-type Ti plasmid, conjugation and octopine utilization were induced by noroctopine. These results are best explained by the activity of a common regulatory gene. In a second step, using homo-octopine, mutants were isolated with lesions preventing the utilization of octopine. All mutations were plasmid borne and did not prevent the induction of tumors. Plasmids of two isolates were characterized by large deletions resulting in a decreased virulence and the absence of octopine in the tumor. With a plasmid carrying an inserted transposon Tn1, a significant number of strains were isolated which were unable both utilize octopine and to transfer the Ti plasmid. This suggests that there may be another common factor--presumably positive--between these traits. Transfer-negative mutants were still virulent. This seems to exclude a role for the conjugative transfer during the process of plant tumor induction. A way to test octopine oxidase by the use of permeable cells is described.


Isolation and characterization of Caulobacter crescentus flagellar hooks.

The basal hook structure of the flagellar organelle Caulobacter crescentus was isolated from release flagella. Hook preparations contained a single major proteins species of 73,000 molecular weight and proteins in smaller amounts that may be minor hook components. Hooks isolated from C. crescents CB13B1a and CB15 were immunologically cross-reactive.


Potentiometric titration of the high- and low-potential 4Fe-4S* centers of Azotobacter vinelandii ferredoxin I.

The high-potential 4Fe-4S* center ofAzotobacter vinelandii ferredoxin I has been titrated potentiometrically by a reductive procedure. The absorbance decrease at 510 nm accompanying the reduction of the high-potential center titrated with an Em of 320 mV (n = 1). The low-potential 4Fe-4S* center was titrated by using the absorbance decrease at 410 nm to monitor its reduction. This center exhibited an Em of -424 mV (n = 1).


Comparison of lipids from Spiroplasma citri and corn stunt spiroplasma.

The qualitative lipid composition of Spiroplasma citri and corn stunt spiroplasma is identical. Small amounts of acylated glucose and steryl glucoside were found.


Dosage schedule and plasma levels of doxepin and desmethyldoxepin.

Plasma levels of doxepin and its metabolite desmethyldoxepin were determined in 7 depressed patients treated with doxepin hydrochloride in 3 divided doses at 1000, 1600, and 2200 hours (t.i.d.), and repeated after changing the dosage schedule to a single daily bedtime (h.s.) dose at 2200 hours. Doxepin and its metabolite were measured at 9000, 1200, 1500, and 1800 hours. None of the individual patients showed clinically significant changes in their plasma concentration of tricyclic antidepressant on the 2 dosage schedules. No difference in the clinical condition of the patients was detected on the 2 dosage schedules using the Zung Self Rating Depression Scale, however patients experienced more morning sedation while on the single h.s. dosage. This study provides pharmacological support for the prescription of doxepin on a once daily basis.


A star-struck service: impact of the admission of a celebrity to an inpatient unit.

The celebrity shares features of the VIP or special patient. The celebrity, however, who refuses to remain anonymous within a hospital setting, often evokes more intense and even specific inappropriate reactions in those around him. His presence creates what the authors call the "star-struck phenomenon." The authors discuss the dynamics that produce this phenomenon.


Family history of alcoholism in patients with chronic fatigue.

Persistent fatigue is a common functional physical complaint. This study reports a possible relationship between parental alcoholism or advanced parental age at the patient's birth and the functional fatigue syndrome.


Dialysis, depression and antidepressants.

The treatment of depression occurring in patients undergoing chronic renal dialysis is discussed. This study was conducted on a Dialysis Unit where 10 Travenol dialyzers of the recycling single by-pass type are used to maintain a total of 22 patients. Imipramine in the usual dosage range and schedule was used when an antidepressant was indicated.


Suicide and female aggression: a contemporary analysis of anomic suicide.

Much has been said about the combined efforts of civil disorder and the emergence of the Women's Right Movement during the mid 1960s-early 1970s, suggesting that perhaps now females would rebel against their traditional subordinate/passive role and aspire to more assertive positions within society. Since social disruption and anomic suicide are closely associated, the authors looked at the relationship between suicide and female assertiveness (aggressive suicides). While male/female suicide rates remained proportional over time, female victims, during this period, did manifest a greater degree of aggression than in the past.


Adolescent infectious mononucleosis with psychosis.

This report describes an adolescent with an acute catatonic schizophrenic illness associated with infectious mononucleosis. The literature and clinical evidence supporting a diagnosis of infectious mononucleosis encephalopathy are reviewed. Diagnostic questions in such cases are discussed from a clinical psychiatric perspective. Therapeutic and developmental issues in managing adolescent psychosis of uncertain etiology are explored.


Thrombocytopenia in the absence of leukopenia associated with the use of neuroleptics.

Thrombocytopenia, a fairly uncommon side effect of phenothiazine treatment, usually appears in the presence of a concommitant leukopenia. The authors report 1 patient in whom the illness appeared in the presence of a long known Beta thalassemia but without evidence of alteration in myeloid or lymphoid series. Platelet changes were seen in the presence of a butyrophenone and an aliphatic phenothiazine. A piperazine derivative was used without difficulty.


Agranulocytosis and chlorpromazine.

A case of death associated with chlorpromazine agranulocytosis and the pertinent literature is reviewed. The authors conclude that the condition is cumulative dose related. Elderly, sickly Caucasian females are the population at high risk. Daily evaluation of the need for medication, the cumulative dose, and signs of infection and fever can reduce the incidence.


Demonstration of somatomedin activity of "multiplication-stimulating activity" in rabbit costal chondrocytes in culture.

Multiplication-stimulating activity (MSA), a substance obtained from conditioned medium of Buffalo rat liver cells, stimulated replication of rabbit costal chondrocytes in culture and their DNA synthesis, sulfation of glycosaminoglycans, protein synthesis, and collagen synthesis. These stimulatory effects of MSA were dose-dependent in serum-free medium, indicating that MSA has intrinsic somatomedin activity. Even after several successive passages, cultured chondrocytes were more responsive to MSA than other organ- and cell-culture systems reported. Therefore, cultured rabbit costal chondrocytes proved a good in vitro system for analysis of somatomedin actions.


Interaction of mucopolysaccharides with glycosaminoglycans on glycosaminoglycan-bound AH-Sepharose 4B.

Chondroitinase C, chondroitinase AC, heparinase, and heparitinase separated from an extract of Flavobacterium heparinum were subjected to affinity chromatography with glycosaminoglycan-bound AH-Sepharose 4B, previously coated non-covalently with glycosaminoglycan, as the matrix. The results suggested the importance of coating the matrix with glycosaminoglycan in the binding of the enzyme protein to the matrix.


Binding of troponin components to tropomyosin fragments.

Binding abilities of troponin components to two fragments of rabbit skeletal alpha-tropomyosin, one the N-chain (residues 1-189) obtained by specific cleavage at Cys 190 and the other the p-fragment (residues 183-284) of the tryptic product, were investigated by gel electrophoresis. The mixture of the tropomyosin fragments showed a new band of complex with either troponin (T + C) or whole troponin, troponin-(T + I + C), irrespective of the presence of Ca2+ in solution. On the other hand, troponin-T and troponin-(T + I) as well as troponin-C, -I, and -(I + C) had little bindin capacity to the tropomyosin fragments. Thus, troponin-C enhances the binding capacity of troponin-T to tropomyosin fragments. A two-site binding of troponin-T to tropomyosin is proposed.


Tropomyosin inhibits the interaction of F-actin and filamin.

The value of flow birefringence of F-actin was greatly decreased by filamin due to precipitate formation. This precipitate could be dispersed into birefringent filaments by sonication. Tropomyosin inhibited precipitation of F-actin induced by filamin, and no decrease in birefringence occurred when filamin was added to tropomyosin-bound F-actin. Hence it appears that filamin acts on F-actin in non-muscle cells similarly to alpha-actinin.


Water-soluble lipoproteins from yolk granules in sea urchin eggs. I. Isolation and general properties.

Most of the water-soluble lipoproteins in sea urchin eggs (Hemicentrotus pulcherrimus) were localized within yolk granules. Under hypotonic conditions, yolk granules released lipoproteins and a 24S protein species as high molecular weight components; the lipoproteins constituted about 40% of the total materials released. Three yolk lipoproteins(YLP-1, 2, and 3, in order of quantity) were isolated by ultracentrifugation and gel filtration. The hydrated densities of YLP-1, 2, and 3 were 1.027, 1.062, and 1.009 g/cm(3), respectively. YLP-1, 2, and 3 contained glyceride as a major lipid in quantities of 3.1, 1.8, and 4.3 times the amount of each protein, respectively. These lipoproteins contained large amounts of carbohydrate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed four major periodic acid-Schiff (PAS) positive polypeptide bands common to the three lipoproteins. All the constituent polypeptides of the 24S protein were also PAS positive. Electron microscopy of negatively stained YLP-1, 2, and 3 revealed the average diameters to be 36, 29, and 48nm, respectively. The 24S protein appeared to be cylindrical in shape with average exterior dimensions of 10--20 nm. Thin-section micrographs showed that yolk granules are packed with particles around 30 nm in diameter, suggesting that these particles are not the 24S proteins but the lipoprotein particles.


Study on the factors yielding high color in the carbazole reaction with hexuronic acid-containing substances.

The color yields with hexuronic acids and hexuronic acid-containing substances were studied by means of the carbazole method of Bitter and Muir with or without 0.025 M borate. The carbazole-borate to carbazole (CB/C) ratios thus obtained indicated not only the degree of the borate effects on the color yields with these materials but also the anomalous nature of some of these substances in the carbozole reaction. The present data indicate that the high color yield with heparin in the carbozole reaction may be due to the production of free amino groups from acid-labile sulfamino groups in the early stage of the reaction, resulting in suppression of protonation on the alpha-D-glucosaminidic oxygen atoms attached to the hexuronic acid residues in the molecule. Moreover, it is suggested that the degree of the unusual color yields in the carbazole reaction with glycosaminoglycans is greatly influenced by the anomeric configuration of the hexosaminyl linkages attached to the hexuronic acid residues in these polymers.


Study on calcium transport by sarcoplasmic reticulum vesicles using fluorescence probes.

Fluorescence changes of 1-anilino-8-naphthalenesulfonic acid and 3,3'-dipropyl-2,2'-thiadicarbocyanine during Ca transport by sarcoplasmic reticulum vesicles were studied. The fluorescence of both probes is enhanced corresponding to the rapid initial Ca uptake. The enhancement could be interpreted in terms of increased binding of the former (anionic) dye and decreased binding of the latter (cationic) dye to the vesicles, suggesting a change in the surface charge of the membranes associated with Ca transport. Under limited conditions where Na, K, and Mg were not added exogenously, Ca could be transported without concomitant counter-transport of these cations.


The determination of molecular weights of Streptomyces subtilisin inhibitor and the complex of Streptomyces subtilisin inhibitor and subtilisin BPN' by sedimentation equilibrium.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.


Separation and purification of phospholipid exchange proteins in rat small intestinal mucosa.

The cytosol fraction of rat small intestinal mucosa stimulated the transfer of [32P]phosphatidylcholine and [32P]phosphatidylinositol from donor liposomes to acceptor liposomes. The proteins which catalyzed the exchanges were separated into three fractions by gel filtration on a Sephadex G-75 column and chromatography on DEAE-cellulose and CM-cellulose. One of the fractions was purified 340-fold and stimulated phosphatidylcholine exchange but not phosphatidylinositol exchange. The other two fractions were active in the stimulation of phosphatidylcholine exchange as well as phosphatidylinositol exchange. These two fractions were purified 35-fold and 44-fold over the cytosol fraction with respect to the phosphatidylinositol exchange activity.


Crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), at 4 A resolution.

The crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), which strongly inhibits bacterial alkaline proteinases specifically, was determined at 4 A resolution using four heavy-atom derivatives. The SSI molecule can be described as an ellipsoid of about 30 X 40 X 65 A composed of two identical subunits each having dimensions of about 35 X 25 X 40 A and a molecular weight of 11,483. The subunit has an extensive beta-sheet structure, but no long alpha-helices are present. Based on the binding sites of platinum reagents known to form coordination complexes with methionine, it is speculated that the P1 residue, Met 73, of the reactive site is at the protruding edge of the subunit. At the subunit-subunit interface, a beta-sheet of one subunit is stacked on top of the corresponding beta-sheet of the other subunit.


"Thermal stability" maps for several double-stranded DNA fragments of known sequence.

The origin of cooperatively melting regions in DNA, which appear as fine structures in the optical melting profile, has been examined for DNA fragments of known base sequences from bacteriophages phiX174 and fd. Thermal stability maps, which indicate the states of base pairs along these DNA strands, were constructed within the established theoretical framework using the parameters which best reproduce the melting profiles obtained by high temperature resolution experiments. By comparing these stability maps with genetic maps, it was found that several cooperatively melting regions which span several hundred bases have some correlation with the gene locations.


Age-related changes in the content of the collagen crosslink, pyridinoline.

Pyridinoline is a crosslinking amino acid of collagen fibers. The age-related changes in the content of pyridinoline were followed for collagens from human and rat costal cartilage and Achilles tendon. The pyridinoline content of the collagens in fetal or newborn animals was very low and increased markedly with growth of the animals. In rat tissues, the pyridinoline content continued to increase after the animal had reached maturity. On the other hand, in human tissues, it began to decrease after about 30 years of age. Pyridinoline may serve as an interesting index for the aging of connective tissues.


Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis.

Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5 X 10(7) and 1.0 X 10(8) cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions or freshly isolated cells and cultured cells were compared in this medium. In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated. These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.


An osmotic effect operative in frontal gel chromatography.

The osmotic effect operative in frontal gel chromatography was quantitatively studied. When mixtures of a non-penetrating solute (Kav = 0) and a partially penetrating solute (0 less than Kav less than 1) were subjected to frontal gel chromatography, the latter formed a coextensive concentration gradient across the trailing boundary of the former, leading to the formation of a second plateau where the concentration exceeded that of the original solution plateau. It was shown that this anomaly, which we have previously predicted, was a direct consequence of osmotic perturbation of the bead size of the Sephadex gel and could be satisfactorily described by an equation based solely on the osmotic distention of the gel beads. Finally, the implications of the osmotic effect in the frontal chromatographic analysis of acceptor-ligand interactions is discussed and a method for correcting this effect is presented.


Isolation of human urinary lysozyme.

For the isolation of human lysozyme from the urine of leukemia patients, a simple method has been established which involves precipitation of urinary proteins by 60% saturation with ammonium sulfate, fractionation of crude lysozyme on Sephadex G-50 and purification by CM-Sepharose chromatography. By this method approximately 60% of the lysozyme in the urine was isolated in a pure state in ten days.


A chromatin-bound neutral protease and its inhibitor in rat peritoneal macrophages.

Rat peritoneal macrophages are known to contain a chymotrypsin-like neutral protease associated with a specific inhibitor. By homogenizing the cells in 0.25 M sucrose (pH 8.0) containing 0.5% Triton X-100, both the protease and the inhibitor were found to be localized in the nuclei, particularly in chromatin. The inhibitory factor in chromatin was then separated from the protease by hydroxylapatite gel chromatography in the presence of 2 M NaCl and 5 M urea. The inhibitor fraction obtained was deproteinized by digestion with Pronase and subsequent extraction with phenol; these treatments did not alter the inhibitory potency. The deproteinized inhibitor fraction had a UV absorption ratio, A280/A260, of 0.61, but it was resistant to digestion with various nucleases, including DNase 1, nuclease P1, and snake venom phosphodiesterase. However, when it was incubated with poly(ADP-ribose) glycohydrolase from calf thymus, the inhibitory potency was markedly decreased. An authentic poly(ADP-ribose), with a mean chain length of approximately 30 ADP-ribose units, produced significant inhibition of the neutral protease isolated from macrophage chromatin. No such inhibition was produced by DNA, single-stranded DNA, RNA, polyadenylate, polyuridylate, polycytidylate, or monomeric ADP-ribose.


Preferential association of newly synthesized H3 and H4 histones with newly replicated DNA.

The assembly of newly synthesized histones into chromatin during replication of MH-134SC cells was studied. Cells pulse-labeled with iododeoxyuridine and [3H]lysine were mixed with an equivalent number of normal cells labeled with [14C]lysine. Nuclease chromatin obtained from pooled cells was fractionated by buoyant-density centrifugation in a gradient containing Metrizamide and 3-iodo-1,2-propanediol. Histones extracted from heavy and normal chromatin regions of the gradient were fractionated by acid-urea gel electrophoresis, and 3H/14C ratios of individual histones were compared. The results showed highly preferential association of newly synthesized H3 and H4 with newly replicated DNA.


D-Glucose anomeric preference of hexokinases in higher animals.

The D-glucose anomeric preference of hexokinases isolated from rat liver, brain, and skeletal muscle, and bovine retina was studied using the glucose-6-phosphate dehydrogenase-NADP system. The ratios of maximum phosphorylation rates of beta-D-glucose to those of alpha-D-glucose were 1.33, 1.46, and 1.54 for hexokinase types I, II, and III from rat liver, 1.45 and 1.63 for type I from rat brain and bovine retina, 1.53 for type II from rat skeletal muscle, and 0.55 (when determined at 5 mM) for type IV (glucokinase) from rat liver, respectively.


Effects of administration of cobalt chloride and cobalt protoporphyrin on delta-aminolevulinate synthase in rat liver.

Cobalt protoporphyrin inhibited the drug-induced increase of delta-aminolevulinate synthase as well as its transfer from the cytosol fraction to the mitochondria in rat liver in a similar way to protoheme. Cobalt chloride given to animals in a large dose exhibited similar effects. Cobalt protoporphyrin was isolated from the liver of rats treated with cobalt chloride. The observed regulatory effects of cobalt chloride with respect to the induction and the intracellular translocation of delta-aminolevulinate synthase may be mediated by cobalt protoporphyrin synthesized in vivo.


Oxygen exchange in silicone rubber capillaries.

Capillaries of 7 and 12.5 mu diameter have been fabricated in silicone rubber. Whole blood treated with heparin has been perfused through these capillaries. Under flowing conditions, no clotting or other clumping effects have been observed and red cells appear to maintain a constant velocity. Oxygen transfer data to and from saline perfusing the 12.5 mu diameter capillaries have been obtained in order to determine how rapidly O2 will permeate the silicone rubber film. The data indicate that the capillaries simulate lung tissue oxygen exchange and will allow for the first time the experimental determination of oxygen exchange kinetics in flowing whole blood.


Instrumentation for monitoring oxygen consumption using a replenishment technique.

An instrument to measure patient oxygen consumption simply and accurately has been built using a replenishment technique. Oxygen is added to the patient's exhaled gas until its oxygen concentration is the same as the oxygen concentration of the inhaled gas. The rate at which oxygen is added is a measure of the patient's oxygen consumption. Bench tests show that oxygen consumption is measured with an accuracy of +/- 2.3 percent and human tests show an accuracy of +/- 5.9 percent. The instrument interfaces easily with a mechanical ventilator and is readily accepted by the clinician.


A novel filler free silicone rubber biomaterial. II. Radiation chemical and physical evaluation.

Hexamethylcyclotrisiloxane was polymerized at 60 degrees C by gamma radiation to yield filler free silicone rubber (FFSR). The G (crosslinking) value of 23.5 was found for the FFSR polymerized for 4 hours, compared to about 2 for the pure polydimethylsiloxane (PDMS). The sol fractions for both FFSR and cross-linked PDMS varied between 5-7%. The FFSR and the cross-linked PDMS both had tensile strength of about 1 kg per cm2 but FFSR was not brittle and could be extended as much as 500% before breaking. It is believed that the marked improvement in physical properties of FFSR is due to the formation of domains of highly cross-linked material which act as "internal filler".


Polymer modification and blood compatibility.

Two procedures have been examined for altering the blood compatibility of copolymers of dimethylaminoethyl methacrylate, where blood compatibility has been measured by the recalcification time of platelet rich plasma. The improvement in blood compatibility of a copolymer of dimethylaminoethyl methacrylate and methyl methacrylate resulting from contact with heparin has been shown to be dependent on both copolymer composition and the method of polymerization. The hydrophilicity of a copolymer of dimethylaminoethyl methacrylate and acrylonitrile has been altered by reaction with ethylene oxide gas. The relationship between time of exposure to ethylene oxide gas. The relationship between time of exposure to ethylene oxide gas and clotting time indicates a dependence of compatibility on copolymer hydrophilicity.


Experimental myringoplasty.

Different materials were compared as implants in performing a myringoplasty on rats immediately after making a total perforation, (autologous fascia, homologous tympanic membrane, homologous collagen, heterologous collagen, heterologous amnion and proplast). As a reference one group of rats had no myringoplasty done after perforating their drums. The animals were examined regularly, then killed after 40 and 64 weeks. Macroscopically and microscopically proplast and homologous collagen were very inferior to homograft tympanic membrane and autologous fascia. The best results were obtained both macroscopically and histologically with heterologous collagen and heterologous amnion.


Intra-articular implant of filamentous carbon fibre in the experimental animal.

The effects of filling drilled holes and articular cartilage grooves in rabbit femora with 9 micrometer diameter filamentous carbon fibre in vivo were studied. The animals were killed at four weeks and five months and the medial and lateral femoral condyles removed and studied morphologically by gross and scanning electron microscopic examination, and histologically using hematoxylin and eosin, van Gieson, toluidine blue and safranin O staining techniques. Fibrous tissue appeared to surround the carbon fibre bundle and emerge at the surface in large organised bundles, overflowing to cover the surface adjacent to the hole defects. Fibrous tissue surrounds the carbon fibre bundle, and forms a new surface over grooves made in the articular cartilage of rabbits that were kept alive for five months, the tissue within the control grooves filled with disorganised fibrous tissue. There was very little foreign body reaction and the fibrous tissue appeared to grow parallel and surround the carbon fibre bundle.


Aortic valve mechanics--Part I: material properties of natural porcine aortic valves.

A new methodology has been developed whereby the inhomogeneous and anisotropic aspects of an irregularly surfaced organ component have been incorporated in establishing the material properties of its tissue. Specifically the direction-dependent post-transition elastic moduli have been calculated from microtensile experiments for the porcine aortic valve leaflets with an average of ECIRC/ERAD = 3.2. Furthermore pressure-strain data have been measured for a grid of points on the surface of a porcine aortic valve and an isostrain map has been developed for P = 80 mm. Hg. These data are used for the stress analysis of porcine aortic valves which is the subject of a companion paper.


Effect of polyurethane morphology on blood coagulation.

A series of polyurethanes based on the hard segment MDI and soft segment PTMG were synthesized. The molecular weight of the PTMG was 730, 1000, and 2000, and ethylene diamine was used as chain extender. The fabrication process was varied so as to achieve the maximum disorder (nonequilibrium state) and maximum order, fully annealed. It was demostrated that the "quenched" non-equilibrium state reduces the rate of activation of the intrinsic systems, factor XII pathway, when compared to the fully annealed state. Platelet attachment is primarily affected by phase separation.


The thrombogenicity of radiation grafted polymers as measured by the vena cava ring test.

Radiation grafted hydrogels on silicone rubber with water contents ranging from 10--90% have been evaluated by the vena cava ring test. All hydrogel grafted materials were found to accumulate less thrombus than ungrafted silicone rubber, and graft level was found to have little effect on thrombus accumulation. Acrylamide grafts demonstrated superior thromboresistance by this test. Poly (2-hydroxyethyl methacrylate-ethyl methacrylate (HEMA/EMA) copolymers were examined along with pure HEMA and EMA grafts. At two weeks implantation, intermediate HEMA/EMA copolymers were found to have the least thrombus accumulation of any of the polymers in this series. EMA grafted rings were highly thrombogenic. The results from these studies indicate that the sensitivity of the vena cava ring test for detecting certain differences in polymer structure is poor. Also, the significance of the vena cava ring test for evaluating materials for use in contact with flowing human blood is questionable, based upon comparisons of results for similar materials tested in other blood compatibility evaluation systems.


In vitro cytotoxicity and in vivo acute responses to some amino acid copolymers and their esters.

The present study was performed in order to establish if there is any correspondence between specific parameters of tissue reaction to implanted biomedical materials and in vitro cytotoxicity. The presence of various types of inflammatory cells and/or necrosis in rats implanted subcutaneously with a series of 42 alpha-amino acid copolymers and their esters was compared with their in vitro toxic effects, as determined by an agar overlay technique. Only necrosis appeared to correlate with reactions scored as strongly positive by tissue culture procedures. Future studies with slightly toxic materials should assess if tissue culture methods could be helpful in predicting other levels of reaction at the polymer-tissue interface.


A model for evaluating the ocular contusion injury potential of propelled objects.

The existing data on experimental ocular contusion were analyzed in terms of a simple mathematical model which predicts the maximum contact force between the eye and the impacting projectile. It was shown that this force was related to the injury producing mechanism, eyeball expansion, which was widely advocated in the literature. The effect of loading rate was also accounted for in the model. A dimensional analysis allowed previous experimental data to be used to generate tolerance curves for ocular injury. The agreement between the prediction of the model and previous subjective opinions of a panel of ophthalmologists was considered to be satisfactory. When the model was used to predict the hazard potential of projectile toys which presently exist in the market place, it was found that the contusive injury producing capability of these products covered the full range from safe to hazardous.


An accurate data collection method for spatial motion using a sonic digitizer.

An accurate collecting method for spatial displacement data utilizing a sonic digitizer which works electronically and acoustically is described. The sequence of the operation of the digitizer system is explained. As an application, the study of elbow flexion-extension is presented. Finally, operational precautions for using this system are discussed.


Isolation of a cross-linked dimer of elastin.

Salt-soluble elastin, isolated by coacervation from extracts of copper-deficient pig aorta, contains a minor protein component separable by gel electrophoresis in 6 M urea. This protein has a molecular weight of 150,000 compared to 75,000 of the major component. The amino acid analyses of both proteins are typical of elastin but the higher molecular weight component has a significantly lower lysine content and lysine-derived cross-links that are lacking from the major component. The proteins were labeled by incubation of the fresh aortic tissue with [14C]lysine. The acid hydrolysate of the borohydride-reduced higher molecular weight protein contained a labeled amino acid eluting at a time identical to standard merodesmosine and a trace of radioactivity corresponding to lysinonorleucine. We conclude that the higher molecular weight protein is a cross-linked dimer of the previously identified soluble elastin.


The amino acid sequence of radioimmunoassayable neurotensin from bovine intestine.

The amino acid sequence of radioimmunoassayable neurotensin, isolated from bovine small intestinal extracts, has been shown to be the same as that of the peptide originally isolated from bovine hypothalamic extracts. This was accomplished by sequence studies on the intact peptide as well as on its chymotryptic and papain-generated fragments. Thus, neurotensin joins the group of biologically active peptides shown to be present in the same molecular form in both brain and intestine.


Erythroglycan, a high molecular weight glycopeptide with the repeating structure [galactosyl-(1 leads to 4)-2-deoxy-2-acetamido-glucosyl(1 leads to 3)] comprising more than one-third of the protein-bound carbohydrate of human erythrocyte stroma.

Glycopeptides of molecualr weight range 7,000 to 11,000, unusual in size and structure, have been partially purified from pronase digests of lipid-free human erythrocyte ghosts; we term this fraction "erythroglycan." These substances comprise about one-third of the galactose and glucosamine of the ghost. Methylation analysis of erythroglycan yields mainly 4-linked glucosamine, 3-linked galactose, and 3,6-linked galactose, along with mannose and fucose derivatives. Hydrazinolysis and nitrous acid deamination degrade erythroglycan to galactosyl-2,5-anhydromannose, indicating a repeating structure of galactosyl, (1 leads to 4)-2deoxy-2-acetamidoglucosyl (1 leads to 3). Digestion with the endo-beta-galactosidase from Escherichia freundii gives only partial cleavage of the erythroglycan, probably because of the arborized structure indicated by the branched galactose. Since sphingosine is not detectable after methanolysis by chemical ionization mass spectrometry, and since amino acids are present, we conclude that these substances are probably glycopeptidic in origin and are not "macroglycolipids." Erythroglycan may have the same type of keratan-like core structure as the long chain blood group glycolipids from human erythrocytes and could be a protein-bound carrier of the ABO determinants.


Resistance of lipophilin, a hydrophobic myelin protein, to denaturation by urea and guanidinium salts.

The influence of urea, guanidinium chloride (GdmCl), and granidinium thiocyanate (GdmSCN) on the solution structure of lipophilin was examined by circular dischroism and fluorescence techniques. According to the CD results, lipophilin retained at least 60% of organized secondary structure in 8 M urea and 6 M GdmCl (measurements were not possible in GdmSCN). This partial denaturation was of a complex, irreversible nature, and was not appreciably enhanced by prolonged incubation (8 days), by heating to 70 degrees C, by disulfide bond reduction, or by pH variation in the range pH 1.5 to 11. Fluorescence studies demonstrated that the tryptophan residues were only slightly perturbed by 8 M and 6 M GdmCl and remained well buried to the permeant quenching agent acrylamide. A greater, but still far from complete, disruption of lipophilin was achieved in 6 M GdmSCN, and fluorescence polarization provided evidence for some form of cooperative structural change induced by increasing concentrations of this reagent. Transfer of the protein from 2-chloroethanol, in which the tryptophan residues are fully exposed, into 6 M GdmSCN by dialysis resulted in reburial of the fluorophores owing to development of tertiary structure. The combined evidence suggests that the extraordinary resistance of lipophilin to these denaturants is due to the presence of an impervious hydrophobic core. In lipophilin and some other membrane-associated proteins, extended sequences of apolar residues might provide the nuclei for such structural domains.


The covalent structure of bovine liver rhodanese. NH2-terminal sequence and partial structural analysis of tryptic peptides from the citraconylated protein.

Nineteen tryptic peptides produced by cleavage at 18 of the 20 arginyl residues in citraconylated S-carboxymethylcysteinyl-rhodanese have been isolated by a combination of gel filtration and high voltage paper electrophoresis. These Tc fragments account for all of the 293 residues in the parent polypeptide and their partial or complete sequences have been determined by automated and manual Edman degradation. In some cases, sequence analyses were completed by degradation of peptides derived by secondary cleavages of the decitraconylated Tc fragments with trypsin, chymotrypsin, or the protease from Staphylococcus aureus. Automated Edman degradation of intact S-carboxymethylcysteinyl-rhodanese was performed for 60 cycles; the information thus obtained permitted the alignment of seven of the Tc fragments and gave the sequence of the first 79 residues in the polypeptide chain. The Tc peptide at the COOH terminus of rhodanese was placed by virtue of the fact that it contained no arginine. Structural analysis of the Tc peptides provided the sequences surrounding all five of the methionyl residues in the enzyme. One of the methionines was found in a 19-residue Tc fragment which also contained the cysteinyl residue essential for catalysis.


The covalent structure of bovine liver rhodanese. Isolation and partial structural analysis of cyanogen bromide fragements and the complete sequence of the enzyme.

Cyanogen bromide fragments from reduced and carboxymethylated rhodanese have been isolated by gel filtration and ion exchange chromatography on columns of Sephadex G-50 and sulfoethyl-Sephadex C-25, respectively. Partial or complete structural analysis of these fragments has permitted the ordering in sequence of all eleven of the nonaligned tryptic peptides from citraconylated, S-carboxymethylcysteinyl-rhodanese and has thus provided the complete covalent structure of the enzyme. Rhodanese is a single polypeptide of 293 residues and the molecule weight calculated from the covalent structural analysis is about 32,900. The cysteinyl residue implicated in the catalytic function of rhodanese is at position 247. In some preparations of the enzyme the NH2-terminal dipeptide Val-His is missing and the sequence begins with the glutamine at position 3. The rhodanese thus obtained contains 291 amino acid residues and possesses full enzymic activity. X-ray crystallographic analysis of rhodanese has shown that the halves of the molecule (Domains I and II) are nearly identical in conformation. Comparative analysis of the sequences in Domains I and II containing residues with conformationally equivalent alpha C atoms has revealed some degree of homology between the halves of the rhodanese polypeptide. Nethertheless, the structural equivalence of the rhodanese domains is reflected much more by their similarity in tertiary structural than by their sequence homology, even when the sequence comparisons are optimized with reference to the crystallographic results.


Active site cysteinyl and arginyl residues of rhodanese. A novel formation of disulfide bonds in the active site promoted by phenylglyoxal.

Chemical modification studies of bovine liver rhodanese have underscored important distinctions between free rhodanese and the catalytic intermediate in which the sulfane atom of the sulfur donor is bound covalently to the enzyme (sulfur-rhodanese). Treatment of free rhodanese with near-stoichiometric quantities of either iodoacetate or phenylglyoxal results in the rapid modification of the essential sulfhydryl group of Cys-247 and the consequent inactivation of the enzyme. Analysis of rate data for the iodoacetate reaction showed that the apparent pK of this group is 7.8 in free rhodanese and 6.7 to 7.0 in complexes of the enzyme with analogs of sulfur donor substrates, in agreement with the previous inference from steady state kinetic observations. Inactivation of free rhodanese by phenylglyoxal in the presence of cyanide was shown to be caused by a novel reaction in which disulfide bonds are formed between Cys-247 and either Cys-254 or Cys-263. In contrast to these results with free rhodanese, the sulfur-substituted enzyme is not inactivated by iodoacetate and is only relatively slowly inactivted by treatment with substantial excesses of phenylglyoxal. The loss of enzyme activity in sulfur-rhodanese does not involve cysteinyl residues but can be correlated with the modification of guanidino groups, notably that of Arg-186, the side chain of which may play a role in substrate binding. These chemical modification studies have implications with respect to the chemical mechanism of rhodanese catalysis and the interpretation of the x-ray crystallographic analysis of this enzyme.


Protein associations and basic protein conformation in the myelin membrane. The use of difluorodinitrobenzene as a cross-linking reagent.

The near-neighbor relationships of proteins in the myelin membrane were examined using dinitrodifluorobenzene and other cross-linking reagents. When intact cat dorsal column or isolated myelin fragments were treated with cross-linking reagents, up to 20% of the myelin basic protein dimerized. The only other cross-linked product formed in the intact cat dorsal column was a heterodimer consisting of myelin basic protein and either the major or minor proteolipid protein. The remaining myelin proteins, including the major proteolipid protein, were cross-linked into very high molecular weight aggregates. In contrast, when the myelin membrane was dipersed in sodium dodecyl sulfate before the addition of cross-linking reagent, all the proteins remained essentially monomeric, with the exception of myelin basic protein which dimerized to some extent. In the absence of cross-linking reagent, it was shown by radioimmunoassay that small amounts of myelin basic protein dimer and the heterodimer were normally present in sodium dodecyl sulfate-polyacrylamide gels. We found no evidence of intramolecular cross-links between the two peptides formed by N-bromosuccinimide cleavage or between the two peptides formed by cyanogen bromide cleavage of the basic protein monomer. The regions of the myelin basic protein molecule involved in dimerization were also determined by similar cleavage of the cross-linked dimer. A rudimentary model for the structure of basic protein dimer in myelin is presented.


The nonpolar peptide segment of cytochrome b5. Binding to phospholipid vesicles and identification of the fluorescent tryptophanyl residue.

The nonpolar peptide segment of cytochrome b5 consisting of the COOH-terminal 43 amino acid residues binds rapidly to dimyristyl lecithin vesicles above the transition temperature of the phospholipid. The binding is complete as indicated by density gradient centrifugation and is accompanied by approximately a 2-fold increase in the fluorescence emission of the protein, and insertion in the bilayer is apparently in an orientation indistinguishable from that of the whole cytochrome b5 molecule. Quantitative reaction of the protein with N-bromosuccinimide destroys the fluorescence of the protein with a stoichiometry which indicates that a single tryptophanyl residue is responsbile for the fluorescence. The fluorescent tryptophanyl residue is one of 2 adjacent residues, Trp-108 or Trp-109, as shown by carboxypeptidase digestion of N-bromosuccinimide-reacted nonpolar peptide.