[Examination of patients with normal and abnormal voice by pneumotachography (author's transl)].

22 patients with normal vocal function and 33 with voice disorders were examined by pneumotachography. The degree of air-flow speed during phonation, the volume of air during phonation, and the duration of phonation were studied in 17 dysphonic patients with complete closure of the glottis, 16 patients with incomplete glottic closure, and 19 euphonic (control) patients. The following results were obtained: 1. The parameters of the study are dependent upon the intensity of phonation. 2. The values of the normal group and the dysphonic patients with complete closure of the glottis during phonation are approximately the same. 3. The values of dysphonic patients with incomplete closure of the glottis are significantly different from the other two groups. According to the results obtained, pneumotachography appears to be a useful method in the evaluation of laryngeal function. Its advantage consists of determining measurable values which are particularly important for followup examinations during treatment. However, the procedure should be used only as part of the complete phoniatric examination.


[Investigations on the incidence of partial agnosia of sounds (author's transl)].

Stammering as a frequent speech disorder in infancy results from many causes. For instance, it is possible that a child with normal peripheral hearing may be unable to distinguish sounds by tone and therefore will repeat them incorrectly. A testing method is presented in which paired pictures are used which differ by the phonologic opposition of some stammered sounds. The results of the test in 100 children who stammer are reported. Further investigations give evidence that other perceptual disorders can be found in supposed agnosias.


Noise attenuation characteristics of the MX-41/AR and the Telephonics circumaural audiometric headsets.

Masking from background noise is often a serious problem when hearing levels are measured at or near the low reference threshold levels specified in the American National Standards Specifications for Audiometers, ANSI S3.6-1969. Since background noise limits for audiometric test rooms are inversely related to the noise reduction characteristics of the earphone/cushion assemblies, it is important that these units have significant and consistent attenuation properties. This paper reports the noise reduction characteristics measured for the conventional TDH-39 earphone mounted in the supra-aural MX-41/AR cushion and for the new TDH-50 Telephonics circumaural headset. The circumaural headset was found to produce an average of about 10 dB more noise attenuation than did a well-adjusted supra-aural device, and the circumaural headset provided much more consistent test-retest noise reduction characteristics. Theoretical aspects of headband force as well as the implications of attenuation measurements for test room recommendations are discussed.


Pure-tone and tympanometric screening: an ideal pair in identification audiometry.

A population of 140 fourth graders participated in a combined audiometric and tympanometric screening project. Pass/fail criteria were established for both subtests. The children were compared between the two tests with the following findings: (1) 52.2% passed both tasks and 5.0% failed both; (2) 36.4% passed the pure-tone task and failed the tympanometric procedure; and (3) 6.4% failed the pure-tone observation and passed the tympanometric study. Of the total population, 42.8% were inconsistent for the norms established for the two tasks. Since the ideal goal of a hearing screening task is to locate children with hearing losses and/or medically related hearing problems, the routine use of tympanometric screening as an integral part of a hearing conservation program is proposed.


The effects of informative and non-informative speech on the judgment of most comfortable listening level.

Fifteen normal-hearing males and fifteen normal-hearing females were presented with a portion of speech played in a forward and a backward direction. The speech was presented in an ascending manner, and the subjects were asked to make comfortable listening judgments. Results indicated that most comfortable listening level is independent of the direction of the speech stimulus and thus independent of information content. Results also showed that sex of the listener plays an unimportant role in most comfortable listening level. Overall, the results indicated that most comfortable listening level results from clinical populations will be little influenced by the listener's ability to understand the stimulus message and thus little influenced by the degree of reduction to speech discrimination ability.


Surgical correction of deviated nasal septum and premaxilla in a colt.

A 6-month-old Appaloosa colt had a deviation of the premaxilla and nasal septum as well as a dorsal hump of the nasal bone and maxillomandibular malocclusion. Two surgical procedures were performed 12 weeks apart to correct these anomalies. An intraoral approach and autogenous rib graft were used to correct the malocclusion and deviation of the premaxilla. Osteotomy of the nasal bone and removal of the nasal septum were performed to help correct the deviation and to facilitate free air passage through the nares.


Burst width tracking: brief tone thresholds in the normal ear.

This study presents a unique procedure for determining the critical duration and temporal summation function. With this procedure, duration as a parameter was used to determine the temporal summation characteristics of ten normal ears. An apparatus was constructed that permitted subjects to track threshold by modulating the burst width of a brief tonal stimulus. The apparatus permitted experimenter control of the rate of burst width modulation and interburst interval. The burst width could be continuously varied by the subject from 10 to 800 msec. Asymptotic regression indicated that the data were best fit to a simple power function of the form Y = AX(B). A logarithmic transformation was applied to the obtained durations, and slopes of integration were determined for each frequency. These values ranged between 6.3 and 10.2 dB/log unit time. Differences among frequencies were found to be significant. Critical durations were obtained for each subject, and these values were consistent with previous data indicating negligible temporal summation about 150 to 200 msec.


Congenital hydranencephaly and cerebellar hypoplasia in calves.

Hydranencephaly with marked cerebellar hypoplasia was diagnosed in 4 calves in a 95-cow herd in British Columbia, Canada. Clinically, these cases were similar to 8 earlier cases on the same farm. Affected calves were born weak, had slightly domed foreheads, and were incoordinated and thus unable to suckle their dams; at least 2 had no eye preservation reflex. The condition was reported to have been common in the Fraser River Valley during the 1973/1974 calving season. Serologic investigations of the dams were negative for bluetongue and infectious bovine rhinotracheitis. An environmental rather than inherited cause was postulated.


Auditory temporal acuity of the hearing impaired and its relationship to speech intelligibility.

Auditory temporal acuity (deltat) was measured in 42 hearing-impaired subjects to investigate the hypothesis that it would facilitate the production of intelligible speech. The measure divided the test population into two subgroups with mean speech intelligibility measures of 77.7 and 52.6%, respectively. These results are discussed along with implications relating to hearing loss and the slope of audiogram.


Relief of the occluded ear sensation to improve earmold comfort.

Earmold comfort is an important factor in the overall satisfaction a hearing-impaired person has with a hearing aid system. Recent research into the subjective effects of earmold venting indicates that, for the naive hearing-aid user, the solid mold produces a "closed" and "blocked" sensation that is relieved to a degree, depending on the vent diameter. To improve earmold comfort, all earmolds should be vented. However, there are certain types of hearing loss where conventional venting techniques would be undesirable, in particular, cases of severe to profound hearing loss where venting would not only adversely affect the desired frequency response characteristics of the hearing aid but would also lead to serious acoustic feedback problems. For this reason a simple system has been developed with sintered filters in the vent line, thus allowing all earmolds to be vented to relieve the occluded ear sensation although retaining the acoustic characteristics of the closed earmold.


Monitoring of hearing aids and earmolds in an educational setting.

Classroom hearing aid and earmold inspection of the amplification worn by 109 school-age children was performed by an audiologist. Hearing aids and earmolds were classified as adequate or inadequate depending on whether optimal function of the hearing aids and good fit of the earmolds was found. Over 60% of the children were found to be using malfunctioning hearing aids or poorly fitting earmolds. The large number of children wearing inadequate amplification demonstrates the need for constant hearing aid monitoring and earmold check as part of aural rehabilitation and educational programs for hearing-impaired children. A method for accomplishing hearing aid and earmold check by parents and school personnel is described.


Use of cytology for tumor diagnosis in private veterinary practice.

Cytologic and histologic services were offered without charge to veterinarians in the greater Tulsa area by the Tulsa Registry of Canine and Feline Neoplasms. During the first 3 years of operation, all veterinarians treating dogs or cats used the histologic service and slightly less than one-half used the cytologic service. The number of cytologic accessions increased 77% during the 2nd year and 27% during the 3rd year. For corresponding years, the increase in histologic accessions was 5% and 11%. The 7 hospitals from which 79% to 90% of the cytologic specimens were received were compared with the 8 to 14 hospitals that used cytologic service infrequently. The frequency of use of cytology was not related to number  of veterinarians in the hospital, number of animals seen in the hospital, or number of tissues received by the Registry from the hospital. The order of frequency of the most numerous cytologic specimens was fine-needle aspirations of masses in or beneath the skin, mammary preparations, effusion sediments, fine-needle aspirations from lymph nodes, and urine sediments.


Effects of the visual modality on acoustic reflex magnitude.

Acoustic reflex magnitude for nine normal hearing subjects was measured with eyes opened and closed under three visual modality conditions: (1) in darkness; (2) in light without the ability to focus; and (3) in light with the ability to focus. Reflex magnitude decreased with eyes open for all three visual modality conditions; however, greater magnitude reduction occurred when the eyes were able to focus. Magnitude reduction for the three visual conditions was: 7% in darkness; 11% without visual focusing; and 20% with visual focusing. Additional data presented suggests reflex magnitude changes occur when eyes are held open with visual stimulation as a variable.


Hepatozoon canis infection in dogs: clinical, radiographic, and hematologic findings.

Hepatozoon canis gametocytes were identified in circulating neutrophils of 3 dogs from different parts of Texas. Clinical signs included intermittent fever, stiffness, and inappetence. The dogs had marked neutrophilic leukocytosis (35,000--51,000/mm3). Parasitemia varied from less than 1% to 60% of the circulating neutrophils. Two of the dogs had periosteal new born reaction on the vertebrae, ilia, ribs, and bones of the limbs proximal to the carpus and tarsus. There was no evidence that specific drug therapy was of value, although use of corticosteroids, aspirin, and antimicrobial agents was accompanied by clinical improvement.


Spur cell anemia in a dog.

Marked spiculation of red blood cells was observed in a young dog with severe hepatic disease. The resulting anemia was similar to spur cell anemia associated with hepatocellular disease in man. Though the exact cause of spur cell formation is not known, abnormalities in serum lipids and red cell membrane lipids are likely involved. A peculiar pattern of serum lipoproteins may have been involved in formation of this dog's spur cells.


Hypercalcemia secondary to chronic renal failure in the dog: a report of four cases.

Hypercalcemia occurred in 4 dogs with renal failure. Primary causes of hypercalcemia previously described in the dog (primary hyperparathyroidism, pseudohyperparathyroidism, vitamin D toxicosis) were not identified. Increased concentrations of circulating immunoreactive parathormone were found in 2 dogs, and thyroparathyroidectomy of 1 dog resulted in decreased serum concentrations of that hormone as well as of calcium. The latter observations indicated that hypercalcemia was related to increased parathormone activity, but the possibility of other homeostatic imbalances was not excluded. It was concluded that renal failure should be considered as a primary cause of hypercalcemia, along with other causes previously identified.


Gastrointestinal helminths in dogs from a humane shelter in Indiana.

Between January 1975 and August 1976, the alimentary tracts of 104 euthanatized dogs from a humane shelter in Tippecanoe County, Indiana, were examined to determine the prevalence of gastrointestinal helminths. The survey was restricted to dogs greater than 1 year old. Their average age was estimated at approximately 2 years. Parasitism was detected in 86.5% of the dogs examined. Ancylostoma caninum was found most often (58.7%), followed by Trichuris vulpis (51.9%), Taenia sp (31.7%), Toxocara canis (18.3%), Dipylidium caninum (16.3%), Toxascaris leonina (8.7%), and Physaloptera rara (3.8%). Monospecific infections were found in 22.1% of the dogs, whereas concurrent infections with 2 or more species were found in 64.4% of the dogs. Most commonly encountered were dogs infected with 2 species of parasites (39.4%), of which hookworms plus whipworms were found most often (32.7%). Male dogs had a higher percentage of multiple infections than did female dogs.


New features in the development of the submucosal gland of the respiratory tract.

The development of submucosal glands in the respiratory tract was studied by light and scanning electron microscopy in the rat, fetal dog and fetal sheep. From the results obtained the present concepts about the formation of these glands in man were questioned and an alternative hypothesis proposed. With scanning electron microscopy the development of the submucosal gland was seen to begin with an aggregation of low electron-responsive cells. Within such an aggregate, a pit, several microns in diameter, was formed. This pit was usually surrounded by medium electron-responsive cells possessing primary cilia in the rat, and by low electron-responsive cells in the fetal dog. In the rat medium electron-responsive cells appeared in other areas of the aggregate, preceded by apical elevations on the low electron-responsive cells. Further development in the rat led to a disappearance of the low electron-responsive cells, differentiation of ciliated and brush cells, and enlargement of the gland orifice. With light microscopy it was observed that the initial gland buds in both the rat and fetal sheep contained lumina several microns in size. These have not been reported by previous investigators. The bud extended into the underlying tissue and developed many simple tubules. The lumina of these tubules were consistently larger than the channel close to the epithelial surface. The cells of these tubules were also the first to differentiate into mucous and serous cells. The development of glands in the rat, in contrast to the sheep, began after birth. In the sheep, unlike the rat, the lumina of the developing glands were often filled with acidic mucosubstances, even though the cells of these glands did not stain for such material. Hence it is suggested that this material is derived from the mucin-containing cells of the surface epithelium and is carried into the interior of the developing gland by the fluid present in the respiratory tract during intrauterine life.


Ultrastruct of the hypothalamic neurosecretory nuclei of the dormouse (Eliomys quercinus L.) in the awakening and hibernating states.

The ultrastructure of the chief neurosecretory nuclei, supraoptic, (SON), parventricular, (PVN) and infundibular (IN), of the dormouse (Eliomys quercinus L.) has been studied during active and hibernating states. In the active state all three nuclei contained light, dark and intermediate type neurons. In hibernation the SON showed only a single light type which differed from the light neurons of the active state; the endoplasmic reticulum was vacuolized and sometimes grouped in 'honey-comb' structures; the cytoplasm contained accumulations of filamentous 'crystalline' material. None of these features occurred in the active state neurons. In the PVN and IN during hibernation both a light and a dark type neuron were present. 'Honey-comb' structures were seen in neurons of the PVN during hibernation, but never in those of the IN. Thus specific morphological features in the SON and PVN appear to be associated with the physiological changes of hibernation.


Polynesian mandibles.

Adult Polynesian mandibles are predominantly of the rocker form. Polynesian crania have an open cranial base angle, and a rather large upper facial height in the adult. The mandibular growth rotations necessary for normal occlusion to be maintained in the presence of this cranial morphology lead to development of a particularly small gonial angle. There is an increased 'bowing' of the basal component of the bone. Such 'bowing' leads to a sufficiently posterior displacement of the basal component in the gonial region for the development of a distinct angular process (for muscle attachment) to be unnecessary.


Continuity of electron histochemically demonstrable substances in the basal lamina, the ground substance of the connective tissue and the intercellular spaces of eptihelial cells of rat gingiva.

Rat gingiva was examined in the electron microscope for substances reactive to ruthenium red and lanthanum nitrate. Both reagents produced an electron-dense band in the basal lamina where the lamina lucida could not be distinguished from the lamina densa. There was continuity between this electron-dense material and that in the ground substance of the adjacent connective tissue and the intercellular spaces of the adjacent epithelial cells. It was concluded that the lamina lucida is not a simple fluid space but is occupied by histchemically demonstrable substances, probably proteoglycans, and that there is a continuum of histochemically demonstrable proteoglycan through which metabolites pass between the blood vessels of the connective tissue and the epithelial cells.


The effect of immune lymphocytes on reaggregation of fetal mouse tissues.

Mouse fetal metanephric tissue was disaggregated and allowed to reaggregate in hanging drop culture in the presence of syngeneic or allogeneic immune lymphocytes. Reaggregation was not obviously impaired.


The topography of root fibres within the sciatic nerve trunk of the dog.

The architecture of the fibres in the sciatic nerve of the dog has been analysed by following the degeneration of fibres resulting from division of the individual spinal nerves which contribute to the sciatic nerve. A pattern has been demonstrated which varies in part with the size of the contribution to the sciatic nerve from each of the spinal nerves L6, L7 and S1. The redistribution of the fibres of each spinal nerve to form the various branches of the sciatic nerve is also described, and the significance of these arrangements is discussed.


Ultrastructural evidence concerning the mode of secretion of electron-dense granules by Clara cells.

An ultrastructural study of the secretory activity of non-ciliated bronchiolar epithelial (Clara) cells has been made. Membrane-bound, electron-dense granules were observed in the apical cytoplasm of Clara cells, immediately beneath the terminal web, and within terminal web regions, with their limiting membranes in close association with the plasma membrane of the cell. Granules were also observed in cytoplasmic pockets with varying amounts of their surfaces exposed to the bronchiolar, lumen, or within cytoplasmic projections into the lumen. Granules that were enveloped in a thin, uniform coat of cytoplasm and granules with no associated cytoplasm were both found free in the bronchiolar lumen. The study is consistent with the conclusion that the granules are secretory in nature, being extruded by a merocrine and possibly an apocrine mechanism.


The architecture of cancellous bone.

The architecture of cancellous bone has been studied in macerated human bones. A number of distinct types of architecture can be recognized as follows: Type I consists of a very delicate meshwork of fine rods. Type II is made up of both rods and plates. Sub-type IIa is a meshwork similar to Type I, but a varying proportion of rad are replaced by delicate plates. Sub-type IIb shows the presence of thin but large fenestrated plates with a well marked orientation in preferred planes; these are interconnected by smaller plates and rods. Sub-type IIc is made up of relatively thick and extensive arranged for the most part parallel to one another, the plates being connected to each other by fine rods. Type III is made up entirely of plates. Delicate plates may form a meshwork in which a directional orientation may or may not be apparent (Sub-type IIIa). Elsewhere better defined, larger plates may enclose tubular spaces (Sub-type IIIb). In some areas (where cancellous bone is very dense) small relatively thick plates enclose irregular spaces; the appearance may closely resemble that of a honeycomb when the spaces are small, but elsewhere the spaces may show a definite directional orientation. The wall of the marrow cavity of long bones is seldom smooth. It is characterized by the presence of plates and rods in various configurations. A distinct marrow cavity is seen in the majority of clavicles examined.


Reinnervation of the rat adrenal medulla transplanted in the anterior eye chamber.

Adrenal medulla transplanted to the anterior chamber of the eye was studied by electron microscopy. Transplanted chromaffin cells consisted of noradrenaline cells, adrenaline cells and small granule-containing cells, and they formed long cytoplasmic processes. Some chromaffin cells were directly opposed to the iridial epithelial cells. A few adrenergic and cholinergic ganglion cells were seen in the transplant. Nerve fibres, mostly cholinergic, but a few adrenergic, were seen within satellite cells enclosing chromaffin and ganglion cells. A few synapses were formed on the chromaffin and ganglion cells, though the number of such synapses remained small even 6 months after transplantation. The possible origin of these nerve fibres, and the specificity of reinnervation are discussed.


Changes in the surface of fine structure of choroid plexus epithelium following chronic acetazolamide treatment.

Surface changes in the epithelium of the choroid plexuses of the lateral and third ventricles of rats induced by chronic administration of acetazolamide have been studied by scanning electron microscopy. After 3 weeks atrophic changes were evident, the microvilli and blebs normally seen on the ventricular surface of the cells appeared attenuated, and in extreme cases they disappeared, leaving the cell surface completely denuded. Localized areas of hypertrophy, indicated by secondary spherical budding, were occasionally observed. The atrophic changes accord with the known inhibitory effects of acetazolamide on CSF formation: perhaps the small number of cells undergoing hypertrophy compensate to some extent for the atrophic ones and maintain some CSF secretion.


Fibre numbers and sizes in the inferior alveolar nerve of the cat.

Previous studies with the light microscope of paraffin-embedded, metal-impregnated sections have probably underestimated the narrow fibre components of the inferior alveolar nerve in the cat. In this study the inferior alveolar nerve of a young adult cat was fixed in situ with glutaraldehyde, post-fixed in osmium and embedded in plastic. A 1 micrometer section of the whole nerve, and an ultrathin section of its largest bundle were cut. Montage micrographs were made from both sections, and from tracings the number and size distribution of the nerve fibres were measured. The entire nerve contained 6856 myelinated nerves whose diameters were distributed bimodally with peaks at 3--4 and 9--10 micrometers. This total is higher than all previously published counts of this nerve, the increase being due to the detection of a greater number of narrower myelinated fibres. In the bundle examined electron microscopically 51.4% of the fibres counted were non-myelinated, four times as many as found in previous studies. The number and distribution of myelinated fibres found by electron microscopy was very similar to that found by light microscopy. The size distribution of myelinated nerves was similar in all the bundles examined. Apart from a lower incidence of non-myelinated fibres, the population of the core of the larger bundle was similar to that of the periphery.


Mitotic rate of the rat's thyroid gland during hypertrophy induced by an antithyroid agent (carbimazole).

Female rats (200 g body weight) were either untreated or given carbimazole (0.1 g/100 ml) in their drinking water for up to 12 weeks. The mitotic rate of the thyroid follicular cells was estimated using vincristine sulphate. Measurement of whole-body oxygen consumption showed that the rats were clearly hypothyroid after 1 week of treatment. The mitotic rate rose rapidly from the control value of 3.65% metaphases/day to about 25%/day after 4 days and was maintained at this level for at least 5 weeks of treatment. The changes in mitotic rate were apparently synchronous with the histological changes. The results of earlier studies are reviewed and discussed in relation to recent concepts of cell populations.


Electron microscope observations on the changing relationships between unmyelinated axons and Schwann cells in human fetal nerves.

The superior rectus oculi muscle from human fetuses of 5, 9.2, 12 and 24 cm crown-rump length (equivalent to 10, 12, 15 and 23 weeks respectively) and a finger from a 9.5 cm crown-rump length (12 weeks) human fetus were examined by electron microscopy. Very simple relationships between Schwann cells and large bundles of axons were found in the superior rectus muscle of the 5 cm fetus and in the large nerve bundles of the fetal finger. Absence of collagen fibrils in the vicinity of these nerve bundles was also noted. In the superior rectus muscles of the older fetuses the complexity of the Schwann cell/axon relationship increased dramatically, the effect being to envelop single axons in Schwann cell processes. This was also the case in the smaller elaborate bundles in the fetal finger which lay adjacent to move primitive, simply invested nerve bundles. In the examination of near serial sections of Schwann cell/axon bundles it was found that the Schwann cell processes, originally likened to a curtain hanging in fluted folds and slightly twisted, was also badly torn and tattered into longitudinal strips of uneven length. In near serial sections, dramatic changes have been found in the arrangements of Schwann cells and axons. Axons may be singled out and separately invested by the Schwann cell processes, but only for short distances. Lateral transfer of axons from one nerve bundle to another may occur, and loss of axons is apparently not uncommon. The same axon may be of very different diameters at different points along its length.


The structure of the germinal disc region of the hen's ovarian follicle during the rapid growth phase.

The structure of the ovarian follicle in the region of the germinal disc, which appears as a white plaque at the surface of the oocyte, was examined by electron microscopy and compared with the non-disc region which overlies the yellow yolk mass of the oocyte in the final growth phase. The main differences concerned the granulosa cell layer and the surface layer of the oocyte. In the disc the granulosa cells were less regularly arranged and the spaces between them varied in width. Their mitotic rate was higher than that in the non-disc region, where cell division was seldom observed at maturity. The perivitelline layer was comparatively poorly developed at the periphery of the germinal vesicle in 15 mm follicles, but eventually attained a uniform thickness throughout the follicle. In the intercellular and perivitelline spaces there were smaller amounts of granular material. Marked differences were observed in the ooxyte surface layer. In 15 mm follicles the surface of the germinal disc was thrown into numerous microvilli and some narrow indentations containing macrovilli from the granulosa cells. Coated vesicles, 120 nm diameter, appeared to be invaginating from the oolemma, whereas 70 nm coated vesicles were present in the deeper cytoplasm. In follicles of more than 25 nm diameter these structural conformations were evident only at the periphery of the disc; for the most part the 120 nm coated vesicles were absent, and over the germinal vesicle microvilli were of rare occurrence. On the other hand, the bulk of the oocyte surface was highly convoluted throughout this period of growth, numerous granulosa cell macrovilli extended into deep pouches associated with 300 nm coated vesicles, and the oolemma possessed a coating of fuzzy material. These observations suggest that there is a restricted passage of yolk precursors to the surface of the germinal disc, and that the inability to transport yellow yolk into the disc is related to differences in the oolemmal surface coat and the population of coated vesicles. The surface modifications, as well as the proliferation of the granulosa cells, are likely to be influenced by the presence of the germinal vesicle.


Effects of intra-articularly administered corticosteroids and salicylates on the surface structure of articular cartilage.

This study was designed to evaluate the effects of repeated intra-articular corticosteroid and salicylate injections on the surface structure of articular cartilage in the rabbit, using the scanning electron miscroscope. We have shown that the normal surface of the rabbit's articular cartilage presents shallow depressions and mound-like elevations and that corticosteroid-treated articular cartilage exhibits progressive lesions, including fissuring and fraying. The effects of salicylates require further investigation.


The mean fibre area of the semitendinosus, diaphragm and pectoralis transversus muscles in differing types of horse and dog.

The mean fibre areas of samples from m. semitendinosus, m. diaphragma and m. pectoralis transversus of thoroughbred horses and greyhounds have been compared with those in similar samples from other members of their species. Athletes (greyhounds and thoroughbreds) have larger fibres in the semitendinosus and diaphragm than their fellows. However, the mean area of pectoralis transversus is similar in the two types of animal within each species. During growth the mean fibre areas increase to a greater extent in athletes than in non-athletes. Detraining adult greyhounds for a period of one year did not signficantly alter the fibre area; nor were any sex differences detected. Although the mean fibre area of corresponding muscles is significantly larger in horses than in dogs, the difference is proportionately less than that of their live weights.


Topographical organization of the striatonigral pathway revealed by anterograde and retrograde neuroanatomical tracing techniques.

L-[4,5-3H]leucine was injected stereotaxically into various regions of the rat neostriatum. Light microscopic autoradiographic techniques were used to plot the entire efferent pathways of the neostriatum, and in particular, the projections to the substantia nigra (SN). The terminal distribution of the pathways projecting to the ipsilateral SN was predominantly restricted to the zona reticulata region. The dorsal part of the head of the striatum was found to innervate the anterior and medial portions of the zona reticulata, while the ventral area of the head projected to more posterior regions of the SN. A pathway from the tail of the striatum to the lateral and dorsal parts of SN was also demonstrated. Horseradish peroxidase injections, restricted to different areas of the SN, led to retrograde labelling of neurons in the striatum whose distribution confirmed the topographic organization of the striatonigral pathway demonstrated in the autoradiographic studies.


A new antibiotic, setomimycin, produced by a strain of Streptomyces.

A new antibiotic, setomimycin, was isolated from the culture broth of strain AM-2947, which was identified as Streptomyces pseudovenezuelae. The compound is a weakly acidic substance, and has UV-absorption at 228, 268 and 422 nm and a molecular formula of C34H28O9 (MW 580). It is active against Gram-positive bacteria including Mycobacteria, and has antitumor activity against Sarcoma-180 solid tumor in mice.


New antibiotics from the fungus Epicoccum nigrum. I. Fermentation, isolation and antibacterial properties.

An atmosphere isolate of the fungus Epicoccum nigrum was found to exhibit an activity against Staphylococcus aureus. A more active, non-sporulating variant of this strain was selected. From its fermentation broth, two novel compounds, epicorazines A and B, were isolated by preparative TLC and tested against a series of bacteria.


Isolation and characterization of elasnin, a new human granulocyte elastase inhibitor produced by a strain of Streptomyces.

Elasnin, a new human granulocyte elastase inhibitor, produced by the strain of KM-2753 designated as Streptomyces noboritoensis KM--2753 has been isolated from the fermentation broth by column chromatography on silica gel and neutral alumina. Elasnin is a neutral, colorless, and viscous oil (ND17 = 1.4983, [alpha]18D -0.9 degrees, lambdaEtOHmax 291 nm (epsilon, 7,760) having a molecular formula of C24H40O4 (MW 392) as shown by its elemental analysis and mass spectrum. Elasnin markedly inhibits human granulocyte elastase, but it is almost inactive against pancreatic elastase, chymotrypsin, and trypsin. At 1.3 microgram/ml (3.3 X 10(-6) M), elasnin is 50% inhibitory to human elastase, but it causes 50% inhibition of pancreatic elastase at 30.1 microgram/ml (76.8 X 10(-6) M).


Early intermediates in the biosynthesis of ansamycins. I. Isolation and identification of protorifamycin I.

A mutant strain derived from Nocardia mediterranei N813 was found to accumulate a number of novel ansamycins structurally related to the protostreptovaricins and to rifamycin W. One of the main components of this ansamycin complex, protorifamycin I (8-deoxyrifamycin W), was purified and identified by means of chemical and spectroscopic methods.


High performance liquid chromatography of natural products. II. Direct biological correlation of components in the fermentation broth.

A portion of the eluent, during liquid chromatography of a fermentation broth, is applied directly to a paper strip at the same rate as the recording of the U. V. absorption. The paper is then placed on the agar inoculated with an appropriate test organism. This procedure permits direct correlation of biological activity with corresponding peaks in the U. V.


Mechanism of chloramphenicol resistance in Bacillus badius 211.

Six strains of chloramphenicol (CM)-resistant endospore-forming bacteria, which can grow in the presence of 100 microgram/ml of CM, were isolated and identified as Bacillus badius. Mechanism of CM-resistance in one of the isolated strains, Bacillus badius 211, was investigated. No inactivation of CM was demonstrated when the strain was grown in nutrient broth containing 100 microgram/ml of CM, as evidenced by paper-disc bioassay of CM in the growth medium. In accordance with this result, no CM acetylation activity was demonstrated either with the intact cells or with the crude extracts of the CM-resistant strain. Poly U- and Poly A-directed polyphenylalanine and polylysine syntheses by S--30 preparations of both CM-resistant and CM-sensitive strains of Bacillus badius were almost equally inhibited by CM. From these results, the mechanism of CM resistance in Bacillus badius 211 seems to be due to other unknown mechanism.


Distribution in normal and inflammatory tissue of a new semisynthetic cephalosporin, SK&F 75073.

SK&F 75073 is a new cephalosporin with broad spectrum antibacterial activity. SK&F 75073-14C and cefazolin-35S were administered separately to groups of rats as a single intramuscular dose of 20 mg/kg. Tissues with highest drug levels 15 minutes following dose were as follows: (SK&F 75073/cefazolin levels), kidney - 86/70 microgram/g, liver - 33/22 microgram/g, lung - 29/17 microgram/g, heart - 23/10 microgram/g, adrenal - 13/7 microgram/g. Plasma levels at peak were 134 microgram SK&F 75073/ml (half-life, 1.9 hours) and 72 microgram cefazolin/ml (half-life, 0.75 hours). Dose excreted in 24 hours was: SK&F 75073, urine 66% and feces 27%; cefazolin, urine 96% and feces 2%. Both antibiotics were also administered, at 20 mg/kg, to rats with the carrageenan-induced inflammatory pouches. Exudate from these pouches contained from 2 to 10 times more SK&F 75073 than cefazolin. Radioassay and bioassay of these substances in the exudate gave similar results. Serum protein binding ranged from 96 approximately 98% for SK&F 75073 and 34 approximately 69% for cefazolin. Data indicated that highly protein bound SK&F 75073 enters tissues and tissue fluid to a greater extent than the lesser bound but therapeutically proven antibiotic agent cefazolin.


Instrumental infrared estimation of fat, protein, and lactose in milk: collaborative study.

Thirty-six milks pre-analyzed by accepted standard methods were analyzed with Milko-scan instruments at 6 laboratories. Using pairs comparisons as described by Youden, precision errors for fat were about 0.033% for the first and 0.022% for the second of duplicate tests, indicating a carry-over effect for the first tests. Precision errors for protein were about 0.021% for both tests. Systematic errors for fat were about 0.17% with calibrations based on reference analyses at each laboratory but were reduced to about 0.044% with calibrations by reference analyses from one laboratory. With homogenized milks, systematic errors for fat increased to about 0.07% and were larger at high fat levels. This effect could be minimized by changing the wavelength of maximum transmission for the fat filter. Systematic errors of about 0.067% for protein were reduced to about 0.03% with calibrations based on common reference analyses. Satisfactory calibrations for lactose were obtained with the 2 Milko-scan 203 models with standard errors of estimate of 0.034 and 0.033%. Results for homogenized and unhomogenized milk were different for most instruments, indicating the need to calibrate for them separately.


Sensitive and specific gas-liquid chromatographic-spectrophotometric screening procedure for trace levels of five sulfonamides in liver, kidney, and muscle tissue.

Free and conjugated sulfonamides are extracted from edible animal tissue with acetone. Carbohydrate is precipitated and removed, and the acetone is evaporated. The residue is transferred to a separatory funnel with ethyl ether and 1N HCl. The acid layer is drawn off and a portion of the 1N HCl is screened, using the Bratton-Marshall reaction. If this portion is positive, the remaining portion is buffered to pH 6.5 and extracted with methylene chloride. The residue is methylated with diazomethane and then acylated with pentafluoropropionic anhydride. The resulting derivatives are detected by gas-liquid chromatography, using electron capture (63Ni) detection and a column packed with 3% OV-17 on Gas-Chrom Q. This method has been validated by recovering sulfathiazole, sulfachloropyrazine, sulfamethazine, sulfadimethoxine, and sulfabromomethazine from liver, kidney, and muscle at levels of 0.1, 0.5, and 1.0 ppm. The 5 sulfonamides were recovered in excess of 60%, with an average mean recovery of 81.5% at the 0.1 ppm level, 79.1% at the 0.5 ppm level, and 76.0% at the 1.0 ppm level.


Modified method for electron capture gas-liquid chromatographic determination of hexestrol residues in bovine tissues.

A gas-liquid chromatographic (GLC) method with electron capture detection was developed for determining hexestrol residues based on a GLC method for diethylstilbestrol residues. The extraction and purification procedures were based on a published procedure. Pentafluoropropionic (PFP) and heptafluorobutyric (HFB) anhydrides and other halogenated compounds were compared as acylation reagents. PFP anhydride was selected because it provided reproducible GLC responses. Of the 3 column packing materials tested, OV-17, OV-210, and QF-1, OV-17 was the most stable under the GLC conditions used. A pH 10.5--10.6 in the purification step gave higher recoveries than pH 10.3 or pH 10.8 and was selected for use. The method is suitable for determining residues of 0.5 ppb. The limit of sensitivity ranges from 0.1 to 0.2 ppb.


Determination of zearalenone in corn by high pressure liquid chromatography and fluorescence detection.

A method is reported for determining zearalenone in corn at levels as low as 10 ng/g. Samples are extracted with chloroform-water and cleaned up by liquid-liquid chromatography, and the zearalenone is detected by a fluorescence detector after separation by reverse phase high pressure liquid chromatography (HPLC). Recoveries of zearalenone added to corn at levels from 10 to 200 ng/g averaged greater than 89%. In addition, a confirmation procedure is described which involves sequential HPLC analysis of the sample and a zearalenone standard, using 4 different excitation wavelengths and comparing fluorescence responses obtained. This method was successfully applied to the analysis of 11 samples of cornmeal; zearalenone was detected in 9 of the samples at levels from 11 to 69 ng/g.


Comparison of sample preparation procedures for colorimetric analysis of nitrite in frankfurters.

Three groups of methods for analyzing nitrite in meat are compared. All methods consist of a sequence of steps, grouped according to initial extraction procedures. In Group 1, the sample is treated with borate followed by HgCl2 or Carrez I, and then Carrez II. In Group 2, the sample is diluted with water, heated at 80 degrees C, and analyzed immediately (AOAC) or after addition of either Na2CO3 and FeCl3 or HgCl2. In Group 3, the sample is made alkaline with NH4Cl buffer and then treated with one of the following: activated carbon plus Carrez I and II, alumina cream, or AIK(SO4)2. At each step when the method involved the addition of a chemical, supernates and precipitates (if formed) were analyzed for nitrite by Griess reagent both before and after AOAC digestion. The normally discarded precipitates formed after addition of HgCl2 and Carrez I and II contained bound nitrite that could be detected by AOAC analysis. Except in the AOAC method, HgCl2 improved nitrite analysis. Results by AOAC analysis were 3 to 300% higher than those determined after addition of any chemical or combination of chemicals. Spiked meat samples could not be used in comparing nitrite analysis methods because results were misleading. Acid meat samples, such as fermented sausages, required neutralization before AOAC analysis.


Liquid chromatographic determination of lasalocid in premixes.

A high pressure liquid chromatographic (HPLC) method has been developed to determine lasalocid in premixes. The method is simple and rapid, requiring an extraction of the drug and separation of insoluble material before HPLC. Elution times are typically less than 10 min per sample. The average recoveries for lasalocid at the 16.5 and 50% levels were 100.2 +/- 2 and 100.0 +/- 2%, respectively. The precision of the method (coefficient of variation = 0.02) compares favorably with that of the approved bioassay (coefficient of variation = 0.06).


Liquid chromatography with fluorescence detection of lasalocid sodium in feeds and premixes.

Lasalocid sodium is extracted directly from feeds and premixes with the mobile phase solution, an aliquot is chromatographed, and the concentration (peak height) of lasalocid sodium is measured by high pressure liquid chromatography with fluorescence detection. Chromatography and detection are completed in about 7 min. The results agree well with those found by the official AOAC fluorometric method, 42.A08--42.A13. The method offers an alternative to the AOAC method and has the capability of processing almost twice as many samples. The average recovery of 4 levels (0.0008, 0.0040, 0.0080, and 0.0120%) of lasalocid sodium from feeds is 100%. Replicate assays of typical poultry mashes and pellets containing about 0.0080% lasalocid sodium yielded coefficients of variation of 2.68--3.35%.


Decomposition in foods (chemical methods). Fluorometric determination of indole in shrimp.

A fluorometric method is described for determining indole in shrimp. The indole is extracted with n-hexane, partitioned into a methanol-saturated sodium chloride solution (9+1), and determined fluorometrically. The detection limit of the method is 0.04 microgram indole/g shrimp. Quantitative analytical data are presented for indole in samples of shrimp determined by the fluorometric method and the AOAC official colorimetric and gas-liquid chromatographic procedures. The correlation coefficient between the data of the fluorometric and colorimetric methods was 0.96. Indole recovered from 25 g samples of fresh shrimp spiked with 6 and 12 microgram indole/25 g ranged from 97 to 106%. The fluorescence response is linear in the range of 1 to 25 microgram indole/100 ml methanol solution and no significant change is noted in solutions kept in the dark 18 hr.


Fluorometric determination of histamine in cheese.

Thirty-one samples of cheese obtained from retail outlets were analyzed for histamine, using an official AOAC fluorometric method. The types of cheese analyzed and the ranges of histamine found were: colby, 0.3--2.8; camembert, 0.4--4.2; cheddar, 1.2--5.8; gouda, 1.3--2.4; provolone, 2.0--23.5; roquefort, 1.0--16.8; mozzarella 1.6--5.0; and swiss, 0.4--250 mg histamine/100 g. Ten of the 12 samples of swiss cheese contained less than 16 mg histamine/100 g. The remaining 2 samples which contained 116 and 250 mg histamine/100 g were judged organoleptically to be of poor quality. An investigation of one processing facility showed that the production of histamine in swiss cheese may have been a result of a hydrogen peroxide/low temperature treatment of the milk supply. Recovery of histamine added to methanol extracts of cheese ranged from 93 to 105%. Histamine content was confirmed by high pressure liquid chromatographic analysis of the methanol extracts.


Determination of streptomycin residues in eggs and stability of residues after cooking.

An assay procedure for streptomycin was developed, using a surfactant-pH 8 buffer extraction, heating at 85 degrees C to eliminate inhibition from lysozyme activity, and centrifuging to remove physical barriers to diffusion. Recoveries of streptomycin from supplemented eggs averaged 42% over the range of 0.33--2.05 microgram streptomycin/g egg. Eggs were supplemented at 3.0, 30.0, and 300 microgram streptomycin/g and subjected to various cooking procedures: frying, poaching, scrambling, and hard boiling. There was little or no loss of activity as a result of the various cooking procedures with the exception of one of the hard boiled varieties where there was a 40% loss only at the 3.0 microgram/g supplementation. Streptomycin residues were quite stable to normal egg preparation procedures.


Determination of neomycin residues in eggs and stability of residues after cooking.

The procedure for neomycin residues used a surfactant to improve extraction, a centrifuge step to eliminate solids that interfere with the diffusion of the antibiotic, and a heat treatment to destroy interfering lysozyme activity. The use of Bcillus stearothermophilus and a 65 degree C incubation yielded a rapid assay with a sensitivity of 0.2 microgram neomycin activity/g egg. Frying eggs caused little or no loss of activity, poaching resulted in 25% loss, and soft boiling and hard boiling caused little or no loss of applied activity. Neomycin residues in eggs were quite stable to normal egg preparation procedures.


Reverse phase high pressure liquid chromatographic determination of vitamin D in fortified milk.

Vitamin D was determined in commercially fortified milk, using reverse phase high pressure liquid chromatography (HPLC). The milk sample was saponified at room temperature and cleaned up on an alumina column to remove cholesterol and carotenes. The sample was then chromatographed in an HPLC system on a 0.26 X 25 cm ODS-HC-Sil-X-1 column using methanol-acetonitrile (1+1) as the mobile phase. For 6 milk samples, the recovery of added vitamin D using this procedure was 92%.


Regulatory agency interaction in the control of drug residues in animal tissue.

The U.S. Department of Agriculture has the responsibility of determining if a method for residues of drugs and pesticides in animal tissues is acceptable for regulatory control, reviewing data on interfering compounds, validating methods, monitoring drug residues in animal and poultry tissues, assigning priority for monitoring drugs and pesticide residues. These responsibilities are carried out in cooperation with the U.S. Food and Drug Administration in the areas of method development, determination of priorities, and punitive action.


Drug residues in animal tissues and their regulatory significance--the Canadian point of view.

Today it is almost impossible to produce food of animal origin which is free from traces of drugs or chemicals. In Canada the problem of drug residues is controlled by a method of assessment of human safety which involves many factors. The toxicity of the drug in laboratory animals or, if possible, in man, is established and a no-effect dose is then estimated. These studies require oral administration of the drug and include acute, subacute, and teratogenicity studies. Depending on these results, chronic reproductive or carcinogenicity studies may be required before a no-effect dose can be estimated. Residue studies must encompass data on metabolism, pharmacokinetics, and depletion studies in edible tissues and for products such as milk and eggs. For veterinary drug residues, we must consider the target food animal with its particular metabolism, tissue disposition, and excretion patterns. The analytical method for residue detection must be acceptable and its sensitivity limits suitable for the drug and its major metabolites.


Control of hormone treatment in animals and residues in meat--regulatory aspects and approaches in methodology.

Although the benefit of using anabolic hormones for fattening purposes under specific situations in animal husbandry (for instance, veal production) has been acknowledged in European countries, the regulatory consequences were quite different from those in the United States. With the exception of the United Kingdom, there were no approvals to use estrogens for other than distinct veterinary purposes. Hence the methodological approaches were rather to control the illegal treatments and imports. One measure is the uterus weight bioassay. Furthermore morphological parameters (prostate test) and estrogen determinations by thin layer chromatography in urine (also from the bladder) were proposed for detection of treated animals. Radioimmunoassays are in development and considered sensitive enough for residue studies in carcasses, perhaps applicable in combination with high pressure liquid chromatography. The re-evaluation of the regulatory situation is in progress in France and Germany and under consideration in the United Kingdom. Guidelines may be the recommendations of the FAO/WHO Symposium 1975 in Rome on anabolic agents in animal production indicating the variety of risks due to different compounds in question and preference to those with low oral activity and defined bio-degradation.


Method for antibiotic determination in animal tissue, as applied to lasalocid.

The development of a tissue residue method for an antibiotic is dependent on the history of the overall problem; the current requirements that must be met for regulatory approval; the chemistry of the compound; its physical/chemical parameters; and its microbiological spectrum for the best possible fit of all of these to ensure a procedure that will work reliably in the originator's laboratory and to the same degree of effectiveness in anyone's laboratory. A note is made of those factors that must be included as controls on the procedure and those experiments that must be included to differentiate matrix from method influences.


Effect of storage and processing on tetracycline residues in meat and bones.

A semiquantitative microbiological screening test for antibiotics, a sensitive and quantitative microbiological assay, and a fluorometric method specific for tetracyclines are described. Using these procedures, tetracycline residues in animals derived from feed can be detected in tissues like organs, muscles, and bones. Meat contaminated with chlortetracycline (CTC) and oxytetracycline (OTC) and stored at +8 degrees C and -22 degrees C showed very little decrease in antibiotic concentration; however, heating above 65 degrees C reduced the tetracycline content in meat. Temperatures above 130 degrees C were necessary to destroy CTC in bones, CTC in bones was insoluble above pH 4. Manufacturing products with contaminated meat reduced the tetracycline content only if heating was involved.


Use of mass spectrometry for confirmation of animal drug residues.

Mass spectrometry is a technique capable of providing unambiguous confirmation of drug residues in edible tissues at trace levels. This has led to an increase in the number of new animal drug applications containing a mass spectrometry procedure as the confirmatory test. Written guidelines do not exist to cover the requirements to be met for acceptance of these procedures. The legal, analytical, and practical requirements to be considered in developing and submitting these procedures will be presented on current requirements and past experiences.


Use of radioimmunoassay for monitoring hormonal residues in edible animal products.

Radioimmunoassay (RIA) is valuable for sensitive and specific routine measurements of hormones in body fluids. The method is also applicable to determine tissue levels of hormones which, according to tracer studies and depending on the compound, are scattered in the lower ng and pg/g range. For practical assay application it must be decided whether the parent compound or a major metabolite should be measured. Both toxicological and practical regulatory aspects have to be considered, but in any case knowledge of the metabolic profile of the compounds is necessary, especially since the specificity of the RIA group analysis is limited. When applying extraction and purification steps, differences between compounds and between tissues have to be considered; generally more difficulties are encountered than with plasma. Validation of the method should first demonstrate no unspecific interference with the antigen-antibody reaction; then the other reliability criteria have to be established. So far RIAs for the quantitation of estrogens, testosterone, progesterone, and trienbolone--a synthetic androgen--in various bovine tissue samples have been developed. The lower limit of sensitivity ranges between 10 and 70 pg/g. Quantitation of physiological levels of testosterone and progesterone is possible, but there are still some problems with estrogens.


Trenbolone acetate: experiences with bound residues in cattle tissues.

Two heifers were implanted with 300 mg of the radiolabeled anabolic steroid, trenbolone acetate (TBA). After a 60 day slaughter and a 60 day removal followed by 76 day slaughter, total 3H-content in various tissues was 0.5--25 ng/g equivalents of TBA. Radioimmunoassay of the tissues showed that only 1--5% of the total residue present was TBA, its main metabolite trenbolone (TBOH), and TBOH glucuronide, plus up to 5% of other organic-soluble material. Of the radioactivity remaining about half was directly water-soluble, and the insoluble residue could be made water-soluble by treatment with the proteolytic enzymes pepsin and trypsin. These 2 portions were purified with Sephadex G-25 to give a low and high molecular weight fraction. Raney nickel reduction of sulfur bonds in either fraction released up to 50% of radioactivity into the organic phase. TLC showed that the latter contained 2 components which had characteristics similar to TBOH and its metabolites, and thus were at least partly drug-related metabolites. In vitro experiments with bovine liver also showed a small but definite protein binding. It is proposed that in dealing with these covalently bound residues, priority be given to the reactive drug intermediate and the type of binding to macromolecules rather than to the presence of the bound residue itself.


Atomic absorption spectrometric determination of arsenic and selenium in offal and fish by hydride generation.

A method for determining As and Se in beef offal and fish was developed. The sample was digested by heating with a mixture of nitric, perchloric, and sulfuric acids. No pre-reduction of As and Se was necessary. Using a simplified generator, the metal hydrides were evolved by reduction with sodium borohydride pellets from sulfuric and hydrochloric acid media. The hydrides were swept by a flow of nitrogen into a nitrogen-hydrogen-entrained air flame. Absolute detection limit of the method was about 6 ng for As and 4 ng for Se, and absolute sensitivity for both metals was estimated to be 5 ng. Effects of the presence of several cations and anions in the matrix were investigated and some were found to have a suppressive effect on the atomic absorption signal. The analytical results obtained for samples of NBS No. 1571 Orchard Leaves and NBS No. 1577 Bovine Liver agreed well with certified values.


Mass fragmentographic determination of pentachlorophenol in rainbow trout.

Pentachlorophenol (PCP) is extracted from selected tissues of rainbow trout and converted to pentachloroanisole (PCA) by means of alkylation in the presence of potassium carbonate as a condensing agent. Pentachlorophenetole (PPT) is used as an internal standard. The electron impact mass fragmentography is carried out by monitoring m/e 280 (PCA), 294 (PPT), and 237. A plot of peak height vs. concentration is linear from 0.03 to 4.0 ng. Accuracy and precision of the method is +/-2% (N - 36). The results are accurate enough to warrant the use of this method for determining PCP in fish, as well as for monitoring PCP from various environmental sources. In addition, this technique provides a simple and rapid method by which a mixture of PCA and PCP can be determined simultaneously.


Cell envelope associations of Aquaspirillum serpens flagella.

Specific regions of the cell envelope associated with the flagellar basal complex of the gram-negative bacterium Aquaspirillum (Spirillum) serpens were identified by studying each of the envelope layers: outer membrane, mucopeptide, and plasma membrane. The outer membrane around the flagella insertion site was differentiated by concentric membrane rings and central perforations surrounded by a closely set collar. The perforations in both the outer membrane and the isolated mucopeptide layer were of a size accomodating the central rod of the basal complex but smaller than either the L or the P disks. The P disk of the complex may lie between the mucopeptide and the outer membrane. Electron microscopy of intact, spheroplasted, or autolyzed preparations did not adequately resolve the location of the inner pair of disks of the basal complex. Freeze-etching, however, revealed differentiation within the plasma membrane that appeared to be related to the basal complex. The convex fracture face showed depressions which are interpreted as impressions of a disk surrounded by a set of evenly spaced macromolecular studs and containing a central "plug" interpreted as the central rod. In thin sections, blebs, which appear to be associated with the flagellar apparatus, were seen on the cytoplasmic side of the plasma membrane. Superimposing the dimensions of the flagellar basal complex and the spacings of the cell envelope layers and using the position of the L disk within the outer membrane for reference, showed that the S disk might be within and the M disk beneath the plasma membrane. A tentative model was developed for comparison with that based on the structure of the Escherichia coli basal complex.


Carotenogenesis associated with arthrosporulation of Trichophyton mentagrophytes.

Carotenoid pigments were demonstrated in arthrospores of the dermatophyte Trichophyton mentagrophytes but were absent from hyphae and microconidia of this fungus. Incubation at higher temperatures (39 degrees C) allowed arthrosporulation to occur, but essentially no carotenoid was detected in such arthrospores. The carotenoid formation in arthrosporulating T. mentagrophytes did not appear to be either induced or stimulated by light illumination. Mature arthrospores contained the carotenoids phytoene, phytofluene,  zeta-carotene, neurosporene, lycopene, and gamma-carotene and a few minor unidentified carotenoids. These carotenoids were localized within intracellular granules consisting of osmiophilic matrices and complex membranous elements. This is the first demonstration of carotenoid pigments in dermatophytic fungi.


Tumor-inducing (Ti) plasmids of Agrobacterium share extensive regions of DNA homology.

Labeled Ti plasmid DNAs from diverse Agrobacterium strains were hybridized to Southern blots of pTi-B6-806 plasmid DNA digest fragments of known map order. The map location of DNA sequences common to all Ti plasmids was found to be extensive, consistent with the view that Ti plasmids have evolved from a common ancestral plasmid.


Ureotelism and ammonotelism in trypanosomatids.

According to their genera, trypanosomatids excrete urea, ammonia, or both. Species of Herpetomonas and Trypanosoma are ammonotelic. Species of Leishmania, Leptomonas, Crithidia, and Blastocrithidia can be ureotelic, ammonotelic, or both, depending on growth media composition.


Induction and regulation of neuraminidase synthesis in Arthrobacter sialophilus.

A variety of N-acetylneuraminic acid (AcNeu) derivatives and analogs were examined as inducers of the extracellular neuraminidase of Arthrobacter sialophilus. Neuraminidase inductions were primarily studied with tryptone-yeast extract-grown cells after washing and resuspension in a defined replacement medium. The addition of readily metabolizable carbon sources to the latter, such as 0.1% casein hydrolysate, glutamate, or glucose, enhanced enzyme synthesis. Enzyme appearance occurred after a lag in the uptake of inducers, suggesting the participation of a co-inducible transport system. Neuraminidase formation during exponential growth in the presence of AcNeu ceased after depletion of this end product from the medium. It was found, besides AcNeu, that its methyl ester, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid methyl ester are each active inducers, whereas beta-anomers of AcNeu-ketosides are not. These results, in comparison to known enzyme specificity, have revealed significant differences and parallels between the inductive and catalytic processes for neuraminidase. In particular, it would appear that the free carboxylate and oxygenation at C-2 of AcNeu, essential for enzyme catalysis with traditional AcNeu substrates, are not necessary for induction and, furthermore, that transition state analogs can specifically induce this enzyme. The failure to observe catabolite repression in this system is discussed in relation to the intermediary metabolism of the genus Arthrobacter.


Multiple genes coding for octopine-degrading enzymes in Agrobacterium.

Most biotype 2 strains of Agrobacterium tumefaciens and A. radiobacter which utilize nopaline also degrade octopine. In all such strains studied, the ability to degrade octopine did not appear to be transferred to plasmidless recipient cells under conditions of plasmid transfer in which the ability to utilize nopaline was transferred. An octopine-degrading mutant was isolated in a strain cured of its plasmid, suggesting that genes of octopine degradation may have a chromosomal location in some strains. In strains in which octopine utilization is coded by plasmid genes, octopine degradation was always inducible, whereas in strains which degrade both octopine and nopaline, octopine utilization was constitutive although nopaline degradation was inducible. When plasmids coding for octopine-utilizing ability were transformed into a strain containing either a nopaline- or null-type plasmid, transformants able to degrade octopine were either not observed or were unstable upon purification. All of these data suggest that plasmids associated with virulence are incompatible with one another, and therefore imply that the major groups of plasmids associated with virulence have a common origin.


Dissimilatory reduction of bisulfite by Desulfovibrio vulgaris.

The reduction of bisulfite by Desulfovibrio vulgaris was investigated. Crude extracts reduced bisulfite to sulfide without the formation (detection) of any intermediates such as trithionate or thiosulfate. When the particulate fractions was removed from crude extracts by high-speed centrifugation, the soluble supernatant fraction reduced bisulfite sequentially to trithionate, thiosulfate, and sulfide. Addition of particles or purified membranes to the soluble fraction restored the original activity demonstrated by crude extracts, i.e., reduction of bisulfite to sulfide without the formation of trithionate and/or thiosulfate. By using antiserum directed against bisulfite reductase, the reduction of bisulfite by crude extracts was inhibited. This finding, in addition to several recycling studies of thiosulfate reduction, provided evidence that bisulfite reduction by D. vulgaris operated through the pathway involving trithionate and thiosulfate as intermediates. The role of membranes in this process is discussed.


Three models of family therapy: prevention, crisis treatment or rehabilitation.

Models of intervention at a family level are described as preventive, crisis intervention or rehabilitative. The preventive intervention model suggests that certain stresses produce family disorganization and individual family members may regress to symptoms of disease. Family dysfunction could be avoided through identification of high risk groups and intervention at developmental milestones. The data on early intervention with children has produced the most promising results. Crisis intervention, the second model, suggests that early identification and prompt intervention may avoid the development of more serious disorganization. The rehabilitation model is focused on changing long term patterns of maladaptive behavior. It includes the homeostatsis model, the conflict resolution model and other approaches to long term family therapy.


Unsocialized aggressive boys: a follow-up study.

Twenty-two boys who met criteria for the diagnosis "unsocialized aggressive reaction" and had been admitted to an inpatient unit were followed up 21 months later. Two thirds were improved. Fighting and resistance to discipline were diminished while impulsiveness and similar traits persisted. Boys who were separated from an antisocial parent but lived in a home did better than others.


Alpha-methyldopa in schizophrenia.

The dopamine hypothesis of schizophrenia suggests that there is a functional excess of dopaminergic activity within the brain of schizophrenics. Alpha-methyldopa interferes with dopaminergic transmission in the central nervous system. We report a case of a schizophrenic in whom, after an overdose of alpha-methyldopa, there was a dramatic and apparently complete resolution of psychotic symptomatology, followed after a few weeks by a sudden return of psychosis.


Mother-son folie a deux: a case report.

A case of folie a deux is presented in which the child is the dominant psychotic partner and his mother is the passive partner in whom a paranoid psychosis is induced. The conditions under which folie a deux may occur, the diagnosis, and the treatment of the disorder are discussed.


Haloperidol and thiothixene in the long-term treatment of chronic schizophrenic outpatients in an urban community: social and vocational adjustment.

Social and vocational adjustment are highly important--but frequently overlooked--measures of the effectiveness of psychotropic agents in therapeutic regimens involving chronic schizophrenic outpatients. This 24-week, double-blind study compared the effectiveness of haloperidol and thiothixene in facilitating social and vocational adjustment, as well as in controlling more traditionally studied psychopathological parameters, among 36 urban schizophrenic outpatients. Significant improvement favoring thiothixene was shown on physicians' ratings, ratings by relatives living in the same household as the patient, and patients' self-ratings. Side effects occurred with similar frequency in both drug groups; no patients were required to discontinue therapy because of side effects. There were no clinically significant abnormalities in laboratory values in either group.


Prazepam in the treatment of anxiety: a placebo-controlled multicenter evaluation.

A double-blind randomized multicenter parallel group comparison of prazepam in divided doses vs. placebo was conducted by 15 investigators among 847 patients presenting with anxiety alone or concurrent with other medical illnesses. Study groups were well matched for age, sex and level of pre-treatment symptomatology. Efficacy evaluation by a physician questionnaire included serial assessment of global improvement plus 10 target signs/symptoms during the 2 to 4 weeks of treatment. Prazepam was statistically significantly superior to placebo as shown on final on-treatment scores for global improvement ratio and for the target symptoms of anxiety, tension, irritability/hostility, depressive mood, insomnia and somatization.


General psychiatry in a subspecialty setting.

Reorganization of outpatient services at Western Psychiatric Institute and Clinic identified a small percentage of patients who did not fit into the subspecialty framework. This necessitated the reestablishment of a General Psychiatry Clinic. A review of 100 patients in the Clinic revealed that they represented the most difficult diagnostic and management problems in the system. General Psychiatry in this subspecialty setting has practically become a subspecialty in its own right, rather than fulfilling a more traditional role of providing general care to the least complicated patients.


Doxepin versus imipramine in psychoneurotic depressed patients with sleep disturbance: a double-blind study.

This double-blind randomized study compared the effects of imipramine (48 patients) and doxepin (49 patients) in psychoneurotic depressed patients with sleep disturbance symptoms. Mean doses of 112.7 mg/day for doxepin and 116.7 mg/day for imipramine were administered for a mean period of 26 days. While both drugs proved effective, 24 of 27 analyses of covariance showed imipramine to be superior to doxepin. This superiority was statistically significant for the anxiety/depression factor measured on the Hamilton Depression Scale and for the total score, general neurotic feelings factor, cognitive performance difficulty factor and anger-hostility cluster on the Lepman-Rickels Scale. Mild-to-moderate side effects were prevalent to a comparable degree in both treatment groups.


Some characteristics of peroxisomes in the slime mold, Dictyostelium discoideum.

Some characteristics of peroxisomes in the slime mold, Dictyostelium discoideum were studied biochemically. The slime mold contained only two peroxisomal enzymes, urate oxidase and catalase. Both activities were concentrated highest in the light mitochondrial fraction, while the highest activity of acid phosphatase as a marker of lysosomes was found in the heavy mitochondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was 1.21--1.22 g/ml, and that of lysosomes was approximately 1.21 g/ml. When the light mitochondrial fraction was treated with deoxycholate, major activities of urate oxidase, catalase, and acid phosphatase were solubilized. With development of the slime mold, both activities of urate oxidase and catalse increased at the stationary stage (4--6 h after) to a great extent and also slightly at the aggregation stage (10--12 h), thereafter they decreased gradually. Acid phosphatase remained with moderately high activity till the culmination stage (18--20 h), and then disappeared rapidly. It was found that enhanced activities of peroxisomal enzymes at the stationary stage represented an increase in the particle fraction and supernatant. On the other hand, activities of acid phosphatase at the same stage increased two-fold only in the supernatant, while the activity in the particle fraction was reduced to one-half.


Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum.

A membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and Cl-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2,6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.


Hydrolysis of sugar nucleotides in chicken egg white in response to embryonic development.

The egg white of newly laid chicken egg was found to contain about 45 mumol of UDP-N-acetylgalactosamine 4-sulfate, 34 mumole of GDP-mannose, 6 mumol of UDP-N-acetylhexosamine, and 1 mumol of UDP-N-acetylgalactosamine 4,6-bissulfate per liter. There was no significant difference between infertile and fertile eggs in the initial levels of the sugar nucleotides. During incubation for 4 days, the nucleotide levels in infertile eggs showed little change while those in fertile eggs fell continuously until the complete disappearance of the nucleotides on the fourth day. Initial removal of the blastoderm from fertile eggs resulted in cessation of the reduction in nucleotide levels in the 2- to 4-day period after the operation. The decrease of UDP-N-acetylgalactosamine 4-sulfate was followed by an increase of 1-phospho-N-acetylgalactosamine 4-sulfate then N-acetylgalactosamine 4-sulfate in the egg white. When UDP-N-acetylgalactosamine 4-[35S]sulfate or GDP-[14C]mannose was injected into the egg white of a fertile egg, the main feature of the metabolism of the labeled compounds was the successive hydrolysis of their pyrophosphate and phosphate bonds, with the formation of sugar 1-phosphate and sugar. A significant activity of nucleotide pyrophosphatase was detected in the egg white in newly laid eggs (0-day egg). However, no such activity could be detected in egg-white specimens from 1-, 2-, and 3-day eggs. The results suggest that although the decrease of sugar nucleotides in the first day could be ascribed to the hydrolytic action of the enzyme originally present in the egg white, the decrease in the subsequent 3 days results from a more complex process in which the hydrolysis of the sugar nucleotides is related to the development of the embryo.


Automatic generation of steady state enzyme rate equation and the associated constraint equations.

A computer algorithm is presented to derive initial velocity rate equation for general enzyme raction mechanisms, including sequential as well as complicated random mechanisms. The method is based on the theory of graph and the theory of prime number. In complicated mechanisms, there are many pathways and hence many cycles. The values of the rate constants are constrained according to the principle of detailed balance: the product of rate constants in the clockwise direction of a cycle must equal to the product of the rate constants in the counter-clockwise direction of the same cycle. An algorithm is presented to derive the appropriate constraint equations. These constraint equations are arranged so that when the rate equation is used for estimating rate constants, the resulting rate constants would satisfy the principle of detailed balance automatically.


Dissociation constant of Tetrahymena tubulin-colchicine complex.

Tetrahymena tubulin has a very low affinity for colchicine. The dissociation rate constant (k-1) and association rate constant(k+1) of Tetrahymena tubulin for colchicine were determined in comparison with those of porcine brain tubulin. The k+1 value of Tetrahymena tubulin was about one-twentieth that of porcine brain tubulin, and k-1 of Tetrahymena tubulin was about 680 times greater than that of brain tubulin. The dissociation constant (k-1/k+1) of Tetrahymena tubulin (2.7 X 10(-3) turned out to be ten thousand times greater than that of brain tubulin. This large Kd value explains well the high colchicine concentration requirement reported to block cell division of cilia regeneration of this protozoan.


Enzymes of the glutamate and aspartate synthetic pathways in a glutamate-producing bacterium, Brevibacterium flavum.

Glutamate-auxotrophic mutants lacking phosphoenolpyruvate carboxylase(PC), citrate synthase (CS) or glutamate dehydrogenase (GD), an aspartate auxotroph lacking aspartate aminotransferase (TA), and a glutamate-aspartate double auxotroph lacking both aconitase (AH) and TA were obtained from Brevibacterium flavum No. 2247, a glutamate-producing bacterium. Prototrophic revertants further derived from the CS- and GD-lacking auxotrophs concomitantly recovered the enzyme activities that their parents had lost. These results indicate involvement of the tricarboxylic acid (TCA) cycle and GD in glutamate biosynthesis, that of PC in the biosynthesis of the TCA cycle intermediates and that of TA in aspartate biosynthesis. The CS-deficient mutants accumulated large amounts of acetate and small amounts of pyruvate, aspartate and alanine, while the GD-deficient strains accumulated large amounts of 2-oxo-glutarate and small amounts of citrate. Synthesis of PC was repressed by either glutamate or aspartate and those of CS and GD were repressed by glutamate, whereas those of pyruvate dehydrogenase (PD), AH, and isocitrate dehydrogenase were not affected significantly by glutamate; that of TA was also not affected by aspartate or by glutamate. The specific activities of PD and AH gave peaks during the cellular cultivation, related to the temporary accumulation of their substrates, pyruvate and citrate, respectively. These and previous results on the regulation of the enzymatic activities provide a definite regulatory mechanism for glutamate and aspartate syntheses.


Metabolism of trichothecene mycotoxins. II. Substrate specificity of microsomal deacetylation of trichothecenes.

The substrate specificity of microsomal nonspecific carboxyesterase [EC 3.1.1.1] from rabbit and rat livers was studied in vitro by using seven (A)-type and six (B)-type 12,13-epoxytrichothecene mycotoxins. The C-4 acetyl residues of diacetoxyscirpenol, T-2 toxin, monoacetylnivalenol (fusarenon-X), and diacetylnivalenol were selectively hydrolyzed by the microsomal esterase to yield the corresponding C-4-deacetylated metabolites: monoacetoxyscirpenol, HT-2 toxin, nivalenol, and 15-acetylnivalenol, respectively. The C-3 acetyl group of monoacetyldeoxynivalenol and the C-8 acetyl group of tetraacetoxyscirpen were also deacetylated. Triacetoxyscirpen gave rise to two unidentified metabolites, which may include a C-4-deacetylated product. 8-Hydroxydiacetoxyscirpenol (neosolaniol), HT-2 toxin, acetyl-T-2 toxin and tetraacetylnivalenol were unaffected by this type of hydrolysis. It follow from these results that the C-4 acetyl residue is hydrolyzed by the microsomal carboxyesterase and substituents at C-3 and C-8 contribute to the selective enzymatic hydrolysis of the C-4 acetyl residue of trichothecenes. Kinetic analysis showed that rabbit microsomal esterase possessed a high affinity for (A)-type trichothecenes such as T-2 toxin and diacetoxyscirpenol, and that of rat microsomes possessed a high affinity for (B)-type trichothecenes such as monoacetylnivalenol (fusarenon-X). The significance of this specific deacetylation reaction is discussed in relation to the biological activity of the trichothecene derivatives as revealed by their inhibitory effect on protein synthesis in rabbit reticulocytes.


Studies on the ferredoxin-ferredoxin-NADP reductase complex: kinetic and solvent perturbation studies on the location of sulfhydryl and aromatic amino acid residues.

The molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33,100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined. The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1 mol of ferredoxin was found to react with about 8 mol of p-chloromercuribenzoate in 10 min. Under low ionic strength conditions (1 mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250 nm were 3,640 and 3,690 M-1.S-1, respectively; under high ionic strength conditions (100 mM NaCl), these rate constants were 1,360 and 1,270 M-1.S-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength conditions were 54 and 1,040 M-1.S-1, respectively. By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8 mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.


Experimental validation of self-instructional modules on the topic of donor phlebotomy.

This validation study investigated the effectiveness of an instructional unit on the topic of donor phlebotomy. The unit consisting of 3 modules based on a videotape and 3 booklets covered (1) donor selection, (2) donor arm preparation, (3) donor phlebotomy. A separate-sample pretest-post-test design was used. All 3 modules were found to be instructionally effective as measured by objective referenced tests. Student appraisal data also was collected for the purpose of revising and improving these materials.


Nonglycosylated mouse L cell interferon produced by the action of tunicamycin.

The effect of tunicamycin, a specific inhibitor of protein glycosylation, on interferon production by L cells induced by Newcastle disease virus was studied. The interferon yields in the presence of tunicamycin ranged from 20 to 100% of control in various experiments. The two molecular species of normal L cell interferon with 40,000 and 24,000 daltons were completely eliminated, and a new species of a smaller size (18,000 daltons) was produced. Radiolabeling of interferon with [35S]methionine and [3H]glucosamine added to the induced cultures, as studied by immunoprecipitation and polyacrylamide gel electrophoresis, indicated that the two species of control interferon were both glycoproteins but tunicamycin interferon contained little or no sugar. In support of this was the loss of binding to lectin from Wistaria floribunda. A change in physical property of the molecule due to the absence of sugar was found in decreased heat stability in the presence of sodium dodecyl sulfate, although in its absence no significant difference from control interferon was observed. These results indicate that tunicamycin is an effective agent in producing nonglycosylated but fully active interferon molecules; the sugar moiety appears to be unnecessary for the antiviral activity and for secretion from cells.


Photochemical cross-linking of reovirus genome RNA in situ and inactivation of viral transcriptase.

Reovirus infectivity and core-associated RNA polymerase activity were decreased by irradiation with long wavelength ultraviolet light in the presence of the 4'-substituted psoralen derivatives, 4'-aminomethyl-4,5',8-trimethylpsoralen and 4'-hydroxymethyl-4,5',8-trimethylpsoralen. Monoadduct formation occurred after photoreaction with low psoralen concentrations or brief irradiation times, and the presence of KCl or magnesium acetate had a protective effect. Under the mild reaction conditions in which 1 molecule of 4'-aminomethyl-4,5',8-trimethylpsoralen was bound covalently per 160 to 290 base-pairs, the polymerase activity was decreased by greater than 90%. At higher drug concentrations or longer times of photoreaction of reovirus cores, the viral RNA was extensively cross-linked indicating that the reovirus genome in situ is double-stranded.


Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2.

We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.


Adenylate deaminase. Kinetic and binding studies on the rabbit muscle enzyme.

Kinetic studies with adenylate deaminase have been performed by stopped flow methods at 20 degrees C in 0.01 M imidazole/HCl, pH 6.5. The data were analyzed using either the whole time course of the reaction or the initial portion of the full time course. At low KCl concentrations, activation by the product IMP complicates any interpretation. In the presence of 0.15 M KCl, the results are interpreted in terms of three types of purine nucleotide binding sites: an active site, an inhibitory site which appears to be relatively specific for nucleoside triphosphates, and an activating site which shows relatively little specificity for nucleoside phosphates. Nucleotide binding to the activating site weakens binding to the inhibitory site. Sigmoidal kinetic data observed as a function of AMP in the presence of the inhibitor GTP are interpreted in terms of AMP binding to the activating site and weakening GTP binding. A fragment of myosin, subfragement-2, which has previously been shown to form a tight complex with adenylate deaminase (Ashby, B., and Frieden, C. (1977) J. Biol. Chem. 252, 1869--1875) activates the deaminase reaction only slightly. Complex formation, however, makes the reaction less susceptible to inhibition by GTP, although high levels of this nucleotide will disrupt the complex. In the presence of GTP or GTP plus subfragment-2, hysteretic effects are observed.


Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle.

Current evidence supports similar functions and mechanisms for cardiac sarcoplasmic reticulum (CSR) as for skeletal sarcoplasmic reticulum (SSR). It is thought that the slower relaxation rate of cardiac muscle compared to fast skeletal muscle reflects the lower ATPase activity and calcium transport of CSR. Possible quantitative differences is phosphorylation, dephosphorylation, and calcium transport of the isolated preparations are studied using a quench-flow apparatus. The results show that both CSR and SSR bind calcium tightly in the absence of ATP, and coupling of E approximately P formation and calcium transport occurs in the transient phase of ATP hydrolysis. The rate of phosphorylation (t-1/2 - 10 ms) of sarcoplasmic reticulum (SR) preloaded with calcium is the same for cardiac and skeletal preparations. However, the rates of dissociation of extra vesicular calcium (10 s-1 versus 15 s-1), phosphorylation of calcium-free SR, and dephosphorylation of E approximately P (8 s-1 versus 12 s-1) are lower for CSR than for SSR. By computer simulation, the apparent rate constants associated with the reduced rates of phosphorylation of calcium-free SR were: 12 s-1 for CSR and 63 s-1 for SSR in the presence of high Mg2+. The difference in the rates may be partly responsible for the lower levels of ATPase and calcium transport activity with characterize cardiac muscle preparations.


Cysteine conjugate beta-lyase in rat liver. A novel enzyme catalyzing formation of thiol-containing metabolites of drugs.

A novel enzyme catalyzing cleavage of the thioether linkage in cysteine conjugates of aromatic compounds, such as 2,4-dinitrobenzene and p-bromobenzene, has been purified about 500-fold from rat liver cytosol. Incubation of S-(2,4-dinitrophenyl)cysteine with the enzyme preparation yielded 2,4-dinitrobenzenethiol, pyruvic acid, and NH3 at equimolar ratios, indicating that the thioether cleavage probably proceeds via an alpha,beta elimination reaction. The thiol product was methylated and the methylated derivative 1-methylthio-2,4-dinitrobenzene, was identified by mass spectrometry and proton NMR spectroscopy. The Km value of S-(2,4-dinitrophenyl)cysteine was 0.5 mM at pH 7.4 in phosphate buffer. The enzyme activity was inhibited by hydroxylamine. No cofactor requirement was observed. A combination of the partially purified enzyme and hepatic microsomes that contain thiol methyltransferase (EC 2.1.1.9) converted cysteine conjugates of 2,4-dinitrobenzene and p-bromobenzene to the corresponding methylthio-containing metabolites; S-adenosylmethionine was required. An important role of this novel beta-lyase enzyme in the formation of methylthio-containing metabolites of various drugs is indicated.


Differential effects of estrogen and progesterone on ovalbumin mRNA utilization.

Progesterone treatment of estrogen-primed chicks leads to a shift in the oviduct polysome profile and an increase in the proportion of cytoplasmic RNA which is ovalbumin mRNA. To determine whether the progesterone effect is primarily transcriptional or translational and whether it is separable from estrogen action, rapid estrogen withdrawal by the anti-estrogen tamoxifen was compared in the presence and absence of progesterone. After estrogen stimulation, 24 h of tamoxifen treatment causes ovalbumin synthesis and ovalbumin mRNA levels to fall 10-fold. The proportion of ovalbumin mRNA in the monosome and supernatant fractions of the polysome profile increases; however, these sequences can associate with polysomes if elongation is inhibited by cycloheximide. Progesterone prevents the tamoxifen effects, even if administered 9 h after tamoxifen (at which time ov mRNA has decreased by 30%). The progesterone-induced increase of ovalbumin mRNA (from 0.64% of cytoplasmic RNA to 0.79%) and the increased proportion of ribosomes in polysomes (from 65% to 80%) are thus independent of estrogen action. Twenty-four hours of progesterone plus tamoxifen treatment enhances initiation of translation. However, after prolonged treatments of several days, translation becomes inefficient: ovalbumin synthesis falls by 30% without a coordinate decrease in ovalbumin mRNA, and a significant proportion (15%) of the ovalbumin mRNA becomes localized in monosomes. Thus, optimal maintenance of oviduct mRNA utilization requires the presence of estrogen.


Precursor of egg white ovomucoid. Amino acid sequence of an NH2-terminal extension.

The translation of ovomucoid mRNA in a reticulocyte lysate protein-synthesizing system yields a precursor form which contains an NH2-terminal extension of 23 amino acid residues. Edman degradation of radioactive translation products (pre-ovomucoid) identified the following sequence: formula : (see text), where the initiator methionine (in parentheses) is the only residue cleaved from the NH2 terminus during cell-free synthesis and the vertical line indicates the site at which pre-ovomucoid is cleaved in vivo to yield ovomucoid. The precursor sequence differs from those of two other proteins (pre-lysozyme and pre-conalbumin) secreted by the same cell, but resembles these and other secretory protein "signal peptides" in both length and hydrophobicity. Pre-ovomucoid does not interact with trypsin in the same manner as mature ovomucoid.


Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5 S ribosomal ribonucleic acid.

Rat liver 5 S ribosomal RNA was oxidized with periodate and coupled by its 3' terminus to Sepharose 4B through an adipic acid dihydrazide spacer. The ribosomal proteins that bind to that nucleic acid were isolated by affinity chromatography and identified by electrophoresis in polyacrylamide gels. The eukaryotic 5 S rRNA binding proteins were L6 and L19: small amounts of L7, L23', L27/L27', L35', and L39 also were bound to the affinity column, but whether they associate directly and specifically with 5 S rRNA is not known. The proteins of the 40 S subunit of rat liver ribosomes did not bind to the 5 S rRNA affintiy column, nor did the proteins of either the large or small subparticle of Escherichia coli ribosomes.


Vitamin K dependence of a calcium-binding protein containing gamma-carboxyglutamic acid in chicken bone.

Bone contains a small protein, osteocalcin, rich in the amino acid gamma-carboxyglutamate (gammaCGlu), which is dependent on vitamin K for its synthesis. The vitamin K dependence of osteocalcin biosynthesis has been studied both in the developing chicken embryo and in the skeletons of young chicks. Osteocalcin formation, assessed by quantitation of gammaCGlu in whole bone, begins in the 8-day embryonic skeleton and increases dramatically until hatching. Biosynthesis of gammaCGlu is warfarinsensitive; time- and dose-related inhibition of up to 75% is observed. Vitamin K deficiency reduces bone gammaCGlu concentrations by 50% in 3-week-old chicks, and this is totally corrected by including vitamin K1 in the diet. Feeding the vitamin K antagonist dicumarol decreases the bone gammaCGlu level 80% after 6 weeks, but no corresponding alterations are observed in the calcium and phosphorous content of the bone ash. Osteocalcin purified from bones of dicumoral-fed chicks exhibits several electrophoretically variant undercarboxylated protein species containing 2 and 3 gammaCGlu/57 amino acid residues rather than the normal complement of 4 gammaCGlu/57 residues.


Studies with a homogeneous enzyme from rabbit erythrocytes catalyzing the insertion of guanine into tRNA.

An enzyme that catalyzes a post-transcriptional modification of tRNA, resulting in replacement of a base from tRNA by guanine, has been purified 2600-fold from rabbit erythrocyte cytosol. The purest preparation migrates as a single protein band on polycrylamide gel electrophoresis and the enzymatic activity co-electrophoreses with this protein. The native enzyme has a molecular weight of 104,000 and is dissociated into two subunits of Mr= 60,000 and 43,000. The Km for guanine is 1.5 x 10(-7) M and for a pure guanine-accepting tRNA is 3.3 x 10(-9) M. The amino acid composition of the pure enzyme has been determined. To our knowledge this is the first study in which the molecular characteristics of a pure enzyme capable of modifying an internal position in tRNA has been reported.


Synergistic regulation of phosphorylase a by glucose and caffeine.

Kinetic studies of both liver and muscle phosphorylase a demonstrate that caffeine and glucose inhibit the binding of glucose 1-phosphate to the enzyme in a synergistic competitive and nonexclusive manner. Inhibition studies for numerous other caffeine analogs show that the muscle enzyme has a relaxed specificity for this negative effector. The liver enzyme is more discriminating by preferential binding of methylated oxypurines. Physiological concentrations of AMP and ATP, which affect the enzymic activity at a separate site, prevent glucose from effectively inhibiting the enzyme. The addition of the second synergistic ligand improves the binding of glucose. These data suggest that glucose homeostasis as regulated by phosphorylase may be dependent on a second ligand and that the role of glucose in this physiological process may have been overestimated. A structural rationalization of this synergistic response is discussed with reference to the crystal structure of the muscle enzyme.