Synthesis of trehalose dimycolate (cord factor) by a cell-free system of Mycobacteriumsmegmatis

A 105,000 x g residue fraction of $\text{Mycobacterium}\text{smegmatis}$ contains an enzyme (acyl transferase) that transfers endogenous mycolic acid to trehalose monomycolate to yield trehalose dimycolate. This enzyme activity is stable to repeated freezing and thawing and is unaffected by the antimycobacterial drug, ethambutol. These results show that trehalose monomycolate is a direct precursor of trehalose dimycolate and suggest the presence of activated mycolic acids (acyl donor) in the cell-free system.     

A new purification procedure for Clostridium difficile enterotoxin

$\text{Clostridium difficile}$ produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A₁ = 41,500, A₂ = 16,000) and its pI is 3.5.     

Effect of a peri fluoro substituent on the conformation of dihydrodiol derivatives of polycyclic aromatic hydrocarbons

$\text{Trans}$-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols formed from the metabolism of 7-fluorobenz[a]anthracene by rat liver microsomes were isolated by reversed-phase high performance liquid chromatography. Ultraviolet absorption, mass, and NMR spectral analyses indicated that the 5,6- and 8,9-dihydrodiols were preferentially in quasi-diaxial conformations, whereas the 3,4- and 10,11-dihydrodiols were preferentially in quasi-diequatorial conformations. CPK space-filling models suggest that the quasi-diaxial conformation is primarily the result of electronic repulsion between the fluorine and the $\text{peri}$ hydroxyl oxygen. These findings provide a structural basis in the interpretation of the carcinogenic potencies of some fluorinated polycyclic aromatic hydrocarbons.     

Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus

The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism $\text{Streptomyces hygroscopicus}$, has been cloned in the $\text{Streptomyces}$ vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of $\text{Streptomyces hygroscopicus}$ DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the $\text{Streptomyces}$ vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.     

Rapid invivo metabolism of Leukotriene C3 in the monkey, Macacairus

[5,6,8,9,11,12-³H₆] Leukotriene C₃ (5 μCi) was injected through a catheter into the right atrium of an anesthetized male monkey. Blood samples were drawn from the aorta via a second catheter. The concentration of tritium in blood decreased from 100 nCi/ml after 5 sec to 1 nCi/ml 15 min after injection, suggesting that leukotriene C₃ was rapidly eliminated from the circulation. Chromatographic analyses of radioactive material in blood collected before recirculation had occurred (15 sec after injection) demonstrated that 40% of the radioactive material had been converted into two less polar metabolites. These products had the same chromatographic properties as leukotrienes D₃ and E₃, respectively. The results indicate that leukotriene C₃ is rapidly transformed by monkey lung $\text{in}\text{vivo}$. Two minutes after injection, the component corresponding to leukotriene E₃ was the predominating metabolite in blood.     

Initial membrane reaction in peptidoglycan synthesis. Perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-Butanol

$\text{Phospho}\text{-N-}\text{acetylmuramyl-pentapeptide translocase}$, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. $\text{n-}\text{Butanol}$ was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12–0.18 M), $\text{n-}\text{butanol}$ causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl $\text{diphosphate}\text{-(N}{}^{\mathrm{?}}\text{-}\text{dansyl)pentapeptide}$. Since no change in emission maximum occurs below 22°C in the presence of 0.12 M $\text{n-}\text{butanol}$, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22°C this concentration of $\text{n-}\text{butanol}$ causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22°C. $\text{n-}\text{Butanol}$ also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with $\text{n-}\text{alkanols}$, e.g. $\text{n-}\text{butanol}$, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.     

Membrane fluidity and drug metabolism in liver microsomes of lean, obob and dbdb mice

The thermally-induced changes of a cytochrome P-450 dependent activity (ethoxycoumarin-O-deethylase) and of the fluorescence polarization of diphenylhexatriene were compared in microsomes from lean, $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice. In lean mice, biphasic plots were obtained with break points in the same range of temperature by both methods, whereas, in $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice, no discontinuities were observed. These results may be related to a modified fatty acid composition of microsomal membranes in mutant mice. They exemplify the influence of the lipid environment on the monooxygenase system as also shown by the modified binding constants of cytochrome P-450 towards type II substrates in $\text{db}\text{db}$ mice.     

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica

The liver fluke, $\text{Fasciola hepatica}$, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl₃ augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl₃ inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

An attempt to isolate Brucellaabortus from uterine flushings of brucellosis-reactor donor cattle

Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10⁴$\text{Brucella}$$\text{abortus}$ (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10⁸ organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated $\text{in}$$\text{vitro}$ culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated $\text{B.}$$\text{abortus}$ (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain $\text{B.}$$\text{abortus}$ which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.     

NADH-dependent glutamate synthase and nitrogen metabolism in Neurospora crassa

The GDH (NADPH) mutant strain $\text{am-1}$ of $\text{N. crassa}$ has sizable pools of glutamine and glutamate under ammonium-limited conditions for which requires an elevated glutamine synthetase activity. Glutamine in the pre$\text{s}$ ence of 2-oxoglutarate, stimulated nicotinamide nucleotide oxidation by crude and purified extracts of the $\text{am-1}$ strain and led to a reductant dependent formation of two molecules of glutamate. Aminooxyacetate did not have any effect on the reaction, whereas azaserine inhibited it completely. It is concluded that in $\text{N. crassa}$ glutamine synthetase and glutamate synthase are responsible for the assimilation of low ammonium concentrations.     

Responses of the mussel Mytilusedulis L. to simulated subarctic tide pool conditions

Mussels in subarctic Norway commonly occur in shallow intertidal pools. By living in pools (a habitat rarely inhabited in lower latitudes) they are insulated against low air temperatures but exposed to high salinities (<70°/oo) beneath overlying ice. Mussels avoid exposing their tissues to such high salinities because a shell valve closure response to low temperature operates at about ?1.5°C before ice sheets form and bottom water salinities rise. Shell valve closure coincides with very low cardiac and ciliary activity.     

Carbohydrate binding activity of a lectin-like glycoprotein from stems and leaves of Dolichosbiflorus

A glycoprotein from the stems and leaves of the $\text{Dolichos}$$\text{biflorus}$ plant that cross reacts with antibodies to the seed lectin has been found to bind to affinity columns of blood group A + H substance covalently linked to Sepharose. This binding of the cross reactive material to the affinity resin differs from that of the seed lectin in that it is easily dissociated with 0.15 M NaCl. Affinity electrophoresis using entrapped blood group A + H substance shows that the carbohydrate binding activity of the cross reactive material is weakly inhibited with N-acetyl-D

-galactosamine and N-acetyl-D

-glucosamine. Glucose, mannose and galactose gave no inhibition when tested at concentrations of 50 mM. These data indicate that the specificity of the cross reactive material is somewhat different from the N-acetyl-D

-galactosamine specificity of the seed lectin. The significance of these findings is discussed in relation to the structural similarities of the cross reactive material and the seed lectin.     

Prostaglandin synthesis in rat embryo tissue: The effect of non-steroidal anti-inflammatory drug in vivo and ex vivo

Aspirin and salicylate are well-known but poorly understood teratogens in laboratory animals. Because aspirin inhibits PG synthesis, we systematically examined PG synthesis in rat embryo homogenates, the inhibition of PG synthesis $\text{in vivo}$ and $\text{ex vivo}$ by various non-steroidal anti-inflammatory drugs, and tested the hypothesis that the inhibition of PG synthesis is responsible for aspirin-induced limb defects in rats. We report that embryonic rat homogenates synthesis 6-keto-PGF_1α, PGE, and PGF in large amounts from endogenous substrate, that aspirin and other non-steroidal anti-inflammatory drugs inhibit PG synthesis $\text{in vitro}$ but not necessarily $\text{in vivo}$, and that contrary to our original hypothesis, the inhibition of PG synthesis is likely not responsible for aspirin-induced limb defects in rats.     

31P NMR studies of metabolism in Acanthamoebacastellanii: Polyphosphate release from encysted cells

${}^{31}\text{P NMR}$ studies of $\text{Acanthamoeba}\text{castellanii}$ have shown that encysting cells release polyphosphate into the encystment medium. Mature cysts contain low levels of polyphosphate, as do vegetative cells. Young cysts (7 days) show detectable levels of nucleotide diphosphates and triphosphate similar to those observed in vegetative cells. Mature cysts (90 days) show only excreted polyphosphate as well as a component which has a chemical shift of a phosphodiester. The inorganic phosphate peak in the cyst shows that the cyst milieu is liquid-like and that the intracellular environment maintains a pH between 6 and 7.5 in the presence of extracellular values from 4 to 9.     

Naphthylmercaptobenzoquinone,a new inhibitor of photophosphorylation in Rhodospirillumrubrum chromatophores at the level of ubiquinone

Naphthylmercaptobenzoquinone (NMBQ) inhibits photophosphorylation in $\text{Rhodospirillum}$$\text{rubrum}$ chromatophores. The endogenous unsupplemented system is the most sensitive one being 50% inhibited by 0.5 μM NMBQ, whereas 20 μM are required for 50% inhibition of photophosphorylation in the presence of N-methylphenazonium methosulfate or diaminodurene. The inhibition is less effective under argon, especially in the presence of ascorbate, and is reversed on addition of various naphthoquinones, which can also reverse the inhibition of photophosphorylation by dibromothymoquinone (DBMIB). Another quinone analog, dodecylmercaptonaphthoquinone (DMNQ), inhibits endogenous photophosphorylation even more effectively than NMBQ, but its inhibition is not reversed by the added naphthoquinones. It is concluded that NMBQ and DBMIB, but not DMNQ, inhibit photophosphorylation in chromatophoresby acting as antagonists of ubiquinone.     

Regulation of nitrate reductase level in pea: Invivo stability by ammonium

Ammonium, the end-product of nitrate-reduction, causes a marked increase in nitrate-dependent formation of nitrate reductase activity in pea shoot apices. The ammonium effect is mediated via a decrease in the rate of nitrate reductase decay. The increased stability of the enzyme in the presence of ammonium is indirect and depends upon protein synthesis. A regulatory role for ammonium-induced protein(s) is suggested.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.