Preliminary studies of a partial cDNA clone of the Eig17-1 gene from Drosophila melanogaster have shown that it encodes a probable cytochrome P450 of unknown function. To further characterize the Eig17-1 gene product, a full-length cDNA clone was isolated from a late-larval cDNA library and sequenced. Eig17-1 encodes a protein of 538 amino acids. The predicted protein is a cytochrome P450 that has been assigned to a new family, CYP18. The CYP18 protein is most closely related to steroid and xenobiotic metabolizing P450s of family CYP2 (30-33% identity), and to vertebrate steroidogenic P450s of families CYP17 and CYP21 (25-28% identity). Developmental Northern blot analysis revealed five distinct periods of Cyp18 expression during postembryonic development. Each period lasted 12-15 h, and was tightly correlated with reported ecdysteroid pulses in the first, second and third larval instars, at the time of pupariation and in pupae. This pattern of expression is consistent with the known induction of Cyp18 transcription by 20-hydroxyecdysone at the time of pupariation and suggests that ecdysteroids are major regulators of Cyp18 expression throughout postembryonic development. Northern blot analysis of RNA isolated from different prepupal tissues indicates that Cyp18 is differentially expressed in various ecdysteroid-responsive tissues. High Cyp18 expression was observed in body wall and gut while negligible expression was observed in salivary glands and fat body.

The cDNA sequence encoding the complete mature form of the steroidogenic ferredoxin from chicken testis has been determined and the amino acid sequence deduced therefrom has been compared with the sequences of bovine, human and porcine steroidogenic ferredoxins. The chicken sequence is between 84% and 88% identical with those of the other mitochondrial iron-sulfur proteins. Thus, the amino acid structure of steroidogenic ferredoxins which transfer electrons to mitochondrial forms of cytochrome P-450 has been very highly conserved over evolutionary time.

Bovine adrenocortical cells in primary culture were used to examine the trophic effect of ACTH on the induction of the 17α-hydroxylase and C-17,20-lyase activities. The addition of exogenous pregnenolone to bovine adrenal microsomes showed the appearance of 17α-hydroxypregnenolone before the formation of dehydroepiandrosterone. The same sequence of activities was evident in postmitochondrial supernate from bovine adrenocortical cells cultured 36 h in the presence of 1 μM ACTH but not in postmitochondrial supernate from control cells. In another study, bovine adrenocortical cells were cultured for 36 h after which 30μM 17α-hydroxypregnenolone was added to the medium and the incubation continued 1 h; there was a 4-fold increase in androgen content in the media from ACTH-treated cells over controls. Measurement of the 17α-hydroxylase and C-17,20-lyase reactions in postmitochondrial supernate from cells cultured 0–72 h in the presence of ACTH or 1 mM dibutyryl cAMP showed concomitant increases in the two activities and both activities were inhibited by the same compounds known to inhibit 17α-hydroxylase activity. These observations support the concept of the co-induction of 17α-hydroxylase and C-17,20-lyase activities in response to ACTH; results in keeping with previous studies indicating that the two activities are catalyzed by a single gene product, the polypeptide chain P-45017a.

Absorption and velocity of sound were measured in various submicron size perfluorochemical (PFC) emulsions in water of the types considered for use as artificial blood substitutes. The attenuation of sound was measured in the frequency range 1 to 95 MHz with computer-assisted VHF and UHF send-receive apparatus. Concentrated aqueous emulsions of F-decalin, F-tributylamine, and F-phenanthrene stabilized by various surfactants including fluorinated surfactants were studied as well as Fluosol-43 emulsion, a commercially produced emulsion for use as an artificial blood substitute. The attenuation of sound waves in the abovementioned PFC emulsions can be theoretically described by heat conduction losses, viscous drag losses, and scattering of sound. In contrast to the majority of hydrocarbon/water emulsions, in PFC emulsions the contribution to acoustic absorption of the viscous drag losses and heat conduction losses are approximately equal to magnitude, though shifted in frequency. This is a result of the high density of perfluorochemicals. [Work partially supported by Advanced Biosystems Inc.]

Previously reported ultrasonic attenuation results in the frequency range 4 to 50 MHz and theoretical predictions [J. R. Allegra and S. A. Hawley, J. Acoust. Soc. Am. 51, 1545 (1971)] for an emulsion of toluene in water have indicated that the observed excess attenuation in the measured frequency range was largely due to heat conduction losses. In the present paper, ultrasonic absorption measurements on a 20% emulsion of toluene in water in the frequency range 1 to 135 MHz at 25 °C are described. The excess attenuation due to the emulsion globules as a function of frequency was compared with explicit expressions for heat conduction, viscous drag losses, and scattering of sound by emulsion globules. Unlike the previous work, the agreement between theoretically predicted excess attenuation and that observed can only be obtained if a wide distribution of globule sizes was assumed. The assumption of a globule size distribution spanning from 600 to 6000 Å in diameter produced a theoretical curve in good agreement with the observed excess absorption over the entire frequency range. [Work supported by ONR.]

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3β-hydroxysteroid dehydrogenase-isomerase complex (3β-HSD), like the 17α-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17α-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3β-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 μM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3β-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.

Apparent specific compressibility βapp, apparent specific volume φv, and shear viscosity measurements on aqueous solutions of polyethylene glycol (PEG) of 600 average molecular weight have indicated that two water molecules are relatively strongly bound to each ether oxygen. The ultrasonic velocity (2 MHz) versus concentration plot passes through a maximum at a PEG/H2O mole ratio of 1:2. Other work provides evidence that this hydration causes the molecule to take on a helical conformation via a hydrogen bond. The strong decrease of βapp and φv of the PEG with the addition of H2O up to two hydration molecules shows that these water molecules are strongly bound. A further decrease of βapp occurs with the addition of more water, but shows a weaker interaction and no further decrease in φv. Similar experiments were made on aqueous A-B-A type block copolymer, the moiety A being polyethylene glycol, but of chain length too short to form the helical structure. There is still an indication of a feature at the 1:2 hydration point, but not so sharply defined as with the PEG. [Work supported by ONR.]

1.1. Both yellow (M3c VII) and white (LU887 × LU897) microplasmodia of Physarum polycephalum follow a sigmoidal growth pattern; the yellow strain proliferates more rapidly.2.2. The white strain exhibits a rapid increase in wet: dry weight ratio, probably accounted for by glycoprotein slime generation.3.3. The yellow strain accumulates protein more rapidly than the white; a difference in the pattern of energy expenditure for the two strains is suggested.4.4. The glucose content of the culture media decreases linearly from 90 mg/ml to zero, the pH changes from 4.6 to greater than 5.3 and lactic acid remains approximately constant after 6 days of culture.

An increment in sound velocity at 2 MHz has been observed as the emulsion globule size decreases with time in a water, toluene, and polyoxethylene (POE)—polyoxypropylene (POP) block copolymer system. The observed effect depends on the copolymer concentration and is largest for the block copolymer composed of 50% POE–50% POP moieties. A general acoustical treatment taking into account viscous loss in the emulsions globules as well as surrounding liquid and heat conduction across the interface fails to explain the observed velocity increment. Aqueous solutions of the above copolymer shows association effects as evidenced by a break in the sound velocity versus concentration curve at approximately 15 g/l. [Work supported by ONR.]

The adrenal gland of the Mongolian gerbil produces nearly equal amounts of 19-hydroxy- and of 11β-hydroxycorticosteroids (Oliver and Peron, Steroids4, 351 (1964)). The intracellular location of the 19-hydroxylase (OHase) was studied by determining the activities of both the 19- and 11β-OHase enzymes in gerbil adrenal homogenate and subcellular fractions. Adrenal preparations were incubated in vitro in the presence of exogenous steroid precursor, androstenedione (A), testosterone (T), or deoxycorticosterone (DOC), plus a source of reducing equivalents. When adrenal homogenate was incubated with A plus isocitrate, the only two products detected were 19-hydroxy A and 11β-hydroxy A; the two were formed at virtually identical rates. With subcellular fractions, the capacity for 19-hydroxylation coincided with that for 11β-hydroxylation; the highest activities were in adrenal mitochondria. For adrenal mitochondria the two hydroxylation reactions were (1) enhanced by the addition of Ca2+ to incubations containing NADPH, (2) supported by either isocitrate or succinate as a source of reducing equivalents, and (3) present at nearly the same levels of activity. The 19- and 11β-OHase activity in other subcellular fractions appeared to be of mitochondrial origin. The cytosol was found to contain a C-17 oxidoreductase catalyzing the interconversion between A and T in the presence of the appropriate NADP(H) cofactor. It was concluded that the 19-OHase of the gerbil adrenal gland is primarily a mitochondrial enzyme. The localization and activities of both the 19- and 11β-OHases account for the relative amounts of the major corticosteroids found in the blood of the gerbil.

1. Coupled mitochondria were isolated from exponentially growing Physarum polycephalum. 2. Activity of malate dehydrogenase (oxalacetate reduction) was 10.9 mumol/min/mg protein; the apparent Km was 64 microM. 3. The activity of NADP-isocitric dehydrogenase (IDH) was 110 nmol/min/mg with apparent Km of 35 microM. 4. NAD-IDH showed allosteric properties with AMP as a positive modulator. The apparent Km for the unmodulated activity, 2 mM, was decreased to 0.95 mM by 0.13 mM AMP. 5. Succinic dehydrogenase activity was estimated as three times higher than that of alpha-glycerophosphate dehydrogenase. 6. Mitochondria contained significant amounts of phenolic compounds. Protein estimation by the Bradford method is recommended.

SummaryAdrenal mitochondria were obtained from rats subjected to two different crowding regiments in order to test the in vitro capacity for 11 β-hydroxylation (11β-OH) as an index of stress. Compared to controls, 110 cm2 of floor space per rat, crowding at 30 cm2/rat markedly increased 11β-OH. Little change occurred when rats were held at 50 cm2/animal. High levels of 11β-OH persisted 3 weeks after the termination of severe crowding. This apparent failure to reverse crowding stress is viewed in terms of pathologic changes that appear to accompany crowding stress. On the basis of measuring 11 β-OH capacity, it is possible to differentiate between the stressful effects of the housing conditions.

Activities of flavin-linked alpha-glycerophosphate, succinic, and malate dehydrogenases were studied in the flight muscles of male houseflies of different ages. Enzyme levels are higher in 4, 14, and 21 day old flies as compared to 7 and 18 day old flies. Results of this study indicate that dehydrogenase activity is apparently unrelated to the age-related decline in flight activity of the housefly.

Mitochondria in the fibrillar flight muscles of higher insects are of unusually large size and are reported to undergo age-related changes in size and number. Fibrillar flight muscles of adult male house flies ranging from 1 to 9 days of age were examined by electron microscopy in order to study the mitochondrial changes with age. In longitudinal sections of the muscle from 1-day-old flies mitochondria are elongated, 2 μ or less in length, and lie parallel to the longitudinal axis of the myofibrils. In cross-sectioned muscles of 1-day-old flies, 3–6 ovoid mitochondria surround each myofibril. Several profiles representing mitochondrial fusion are observed in flies 2 days of age. Longitudinal sections of the muscle in 9-day-old flies reveal highly elongated mitochondria ranging up to 15 μ in length. In cross sections, mitochondria form broad sleeve like structures in close apposition with the myofibrils. The bulky mitochondria in 9-day-old flies seem to be formed by the end-to-end, as well as side-to-side, fusion of smaller mitochondria. Histometric comparison indicated that the mitochondria in the flight muscle of 9-day-old flies are significantly larger in size and fewer in number than those of 1-day-old flies. It is suggested that the “giant” mitochondria in the flight muscles of the house fly arise primarily by an age-related fusion of smaller mitochondria.