Minor and trace sterols in marine invertebrates XVII. 1 (24R)-24,26-dimethylcholesta-5,26-dien-3β-01, a new sterol from the sponge Petrosia ficiformis

More than thirty monohydroxy sterols were detected in the marine sponge $\text{Petrosia ficiformis}$, ranging from the ubiquitous 24-norcholesta-5, 22-dien-3β-o1 to the C³⁰ sterol, 24-propylidenecholesterol. A new minor sterol was isolated and shown by spectral analysis and comparison with a synthetic sample to be (24R)-24,26-dimethy1cho1esta-5,26-dien-3β-o1 [26(29)-dehydroap1ysterol] ($\text{12}$).     

Cyclopenta(f)isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid. 3. Interaction of 6-carbamylmethyl-8-methyl-7H-cyclopenta(f)isoquinolin-3(2H)-one with deoxyribonucleic acids and polydeoxyribonucleotides

By the use of space-filling models, a novel compound, 6-carbamylmethyl-8-methyl-7$\text{H}$-cyclopenta[f]isoquinolin-3(2$\text{H}$)-one ($\text{1}$) was devised which would be expected to hydrogen bond specifically to GC pairs in the major groove of the double helix such that (i) the amino group of the cytosine molecule donates a hydrogen bond to the C-3 carbonyl of the isoquinoline moiety and (ii) the amide proton of the side chain donates a hydrogen bond to the N-7 of guanine. From difference spectra studies it was found that $\text{1}$ binds to native calf thymus DNA better than to denatured DNA; $\text{1}$ inhibited RNA synthesis by a DNA-dependent RNA polymerase; and equilibrium dialysis experiments revealed that $\text{1}$ binds to poly(dG).poly(dC), whereas no such binding to poly(dA).poly(dT) was observed.     

The anal sac secretion of the red fox (Vulpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of the lion (Panthera leo)

Phenylacetic, 3-phenylpropionic, $\text{p}$-hydroxyphenylacetic and 3 ($\text{p}$-hydroxyphenyl) propionic acids together with the series of C₂ to C₆ saturated fatty acids previously reported in the anal sac secretion of the red fox ($\text{Vulpes vulpes}$) are identified as constituents of the anal sac secretion of the lion (Panthera leo). All these compounds are also observed in the anal sac secretion of the red fox using gas chromatography. The aerobic microflora of red fox and domestic dog ($\text{Canis familiaris}$) anal sac secretion samples invariably consisted predominantly of $\text{Streptococcus faecium}$ and $\text{Streptococcus faecalis}$. The hypothesis that the secretion volatiles so far identified may be microbiologically produced is examined.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

X-ray analysis of a progesterone-binding protein (uteroglobin): preliminary results

Uteroglobin, which is a progesterone-binding protein of the rabbit uterine secretion, has been crystallized and subjected to X-ray diffraction analysis. Two crystalline forms have been observed: a triclinic one ($\text{P1, Z = 2, a = 36.36 (4)}\text{A}\text{?}\text{, b = 37.40 (4)}\text{A}\text{?}\text{, c = 53.28 (4)}\text{A}\text{?}\text{, α = 104.6 (1) °, β = 97.0 (1) °, γ = 111.3(1) °}$); and an orthorhombic one for which the cell is C-centred with $\text{a = 50.86 (5)}\text{A}\text{?}\text{, b = 52.22 (5)}\text{A}\text{?}\text{, c = 47.28 (5)}\text{A}\text{?}$, space group C222₁, Z = 4. Three isomorphous derivatives have been obtained. The crystals appear suitable for detailed study of the three-dimensional structure of the protein.     

A new site-specific endonuclease from Streptomyces lavendulae (SlaI)

A new restriction-like endonuclease, SlaI, was found and partially purified from Streptomyces lavendulae ATCC8664. This endonuclease cleaved bacteriophage lambda DNA at only one site, and cytosine-substituted bacteriophage T4 DNA at 16 sites. The recognition sequence was determined by using SlaI fragments of cytosine-substituted bacteriophage T4 DNA. The hexanucleotide recognized by SlaI endonuclease was

$\text{5′-}\text{C}\text{?}\text{T}\text{-}\text{C}\text{-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-3′}$

$\text{3′-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-}\text{C}\text{-}\text{A}\text{-↑}\text{C}\text{-5′}$

with the sites of cleavage as indicated by the arrows. Therefore, SlaI endonuclease was an isochizomer of XhoI endonuclease.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Reflex ovulation in light-induced persistent estrus (LLPE) rats: Role of sensory stimuli and the adrenals

Penile intromissions have been thought to be the primary stimulus for reflex ovulation in light-induced persistent estrus (LLPE) rats, even though other stimuli also trigger reflex ovulation. To clarify the nature of these noncoital stimuli, intact (nonadrenalectomized) LLPE rats were briefly exposed to a variety of environmental stimuli, other than intromissions, and checked for ova 19–22 hr later. Summary of results (number of rats ovulating/number of rats tested): (A₁) home cage ($\text{3}\text{10}$); (B₁) home cage + vaginal taping ($\text{2}\text{9}$); (C₁) home cage + male-soiled bedding ($\text{15}\text{28}$); (D₁) novel cage ($\text{2}\text{11}$); (E₁) novel cage + vaginal taping ($\text{2}\text{11}$); (F₁) novel cage + vaginal taping + male-soiled bedding ($\text{9}\text{19}$); (G₁) novel cage + vaginal taping + male-soiled bedding + male mounts without intromissions ($\text{14}\text{26}$). The percentage of LLPE rats that ovulated in the last-mentioned test condition was related to the degree of proceptivity/receptivity of the LLPE females. Eight of eight proceptive LLPE females ovulated, but only $\text{6}\text{18}$ nonproceptive females ovulated. To account for reflex ovulation in the absence of intromission it has been suggested that adrenal progesterone (P) stimulates release of an ovulatory quota of luteinizing hormone. This study demonstrates no significant differences in percentage of LLPE females ovulating in corresponding groups of adrenalectomized (ADX) and adrenal-intact females. Summary of results: A₂ = $\text{0}\text{6}$, B₂ = $\text{5}\text{15}$, C₂ = $\text{4}\text{16}$, D₂ = $\text{2}\text{14}$, E₂ = $\text{5}\text{13}$, F₂ = $\text{7}\text{19}$, G₂ = $\text{10}\text{21}$. Conclusion: (a) Exposure to a factor in male-soiled bedding induces reflex ovulation in a significant proportion of adrenal-intact LLPE animals while exposure to a novel cage and/or vaginal taping does not, (b) penile intromissions are not the primary stimulus for reflex ovulation in intact proceptive LLPE rats, and (c) adrenal P is not required for reflex ovulation after tests with noncoital stimuli.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

The effect of oxygen on CO2 fixation by mesophyll protoplast extracts of C3 and C4 plants.

Oxygen inhibits CO₂ fixation by mesophyll protoplast extracts of the C₃ plants, $\text{Hordeum vulgare}$ and $\text{Triticum aestivum}$, but stimulates the pyruvate induced CO₂ fixation by mesophyll protoplast extracts of the C₄ plants $\text{Digitaria sanguinalis}$ and $\text{Urochloa panicoides}$. The former is reversed by increased levels of bicarbonate, whereas the latter effect is independent of bicarbonate concentration. The results are consistent with the proposal that oxygen inhibits C₃ photosynthesis by competing with CO₂ in the RuDP carboxylase/oxygenase system. The oxygen enhancement of C₄ mesophyll photosynthesis is proposed to be due to pseudocyclic electron flow supplying additional ATP for the CO₂ fixation process.     

A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

A cobalt-porphyrin containing protein has been isolated from the sulfate-reducer $\text{Desulfovibrio desulfuricans}$ (Norway). This violet-colored protein has a molecular weight of approx. 13,000 daltons and contains 1 cobalt atom/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The UV-visible absorption spectrum of the protein exhibiting maxima at 588,418 and 280 nm with a shoulder at 550 nm is characteristic of metalloporphyrin proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio $\text{A}{}_{280}\text{A}{}_{588}$ is 0.54. The protein is reduced by dithionite giving a blue-colored reduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the α band in the opposite direction from 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase from $\text{D.desulfuricans}$ under hydrogen in the presence of cytochrome C₃. The reported data suggest that the redox states of the cobalt center of this new electron carrier correspond to the Co(III) and Co(II) states.     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

The dependence of the conformation of a (1→3)-β-D-glucan on chain-length in alkaline solution

(1→3)-β-D-Glucans of various degrees of polymerization were prepared by degradation of a gel-forming D-glucan with formic acid. The degraded D-glucans were separated into a water-soluble fraction (soluble D-glucan) and an insoluble fraction (insoluble D-glucan). Both D-glucans were further fractionated. The optical rotation including determination of the o.r.d. curves of the fractions and of the original gel-forming D-glucans was measured at various sodium hydroxide concentrations (0–5M). The results indicate that (1→3)-β-D-glucans of $\text{DP}$n below ca. 25 (the soluble D-glucan) took a disordered form in both neutral and alkaline solutions, whereas the D-glucans of higher $\text{DP}$n (the insoluble and the original D-glucans) took an ordered structure in dilute alkaline solution (0.1M). The proportion of ordered structure in the insoluble D-glucan increases with $\text{DP}$n to attain a maximum value at a $\text{DP}$n of around 200; this may be the lower limit of $\text{DP}$n to permit gel formation in neutral media. The formation of complexes with Congo Red in alkaline solutions by the soluble and the insoluble D-glucans supports the same conclusions.     

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β-p-bromobenzoyl ester of diol B was prepared from 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; $\text{a = 9.24}\text{A}\text{?}$, $\text{b = 12.61}\text{A}\text{?}$, $\text{c = 7.03}\text{A}\text{?}$, α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.     

The present status of the role of the Parainfluenza-3 (PI-3) virus in fetal disease of cattle and sheep

The Bovine Parainfluenza-3 (PI-3) virus was isolated and identified from an aborted bovine fetus. The fetal isolate was characterized and found to be similar to the respiratory isolate. $\text{In}$$\text{utero}$ inoculations of bovine fetuses with the PI-3 fetal isolate established fetal pathogenicity, and fetal immune competency. Inoculation of pregnant immune heifers and ewes failed to demonstrate transplacental transmission of virus. Sera from 1500 cows were examined for the presence of PI-3 serum neutralizing (SN) antibody. All serum samples contained PI-3 SN antibody at the 1:2 dilution and greater. Since PI-3 seropositive animals resist transplacental transmission of virus, and since seronegative animals are rarely available, the Bovine Parainfluenza-3 virus is probably not a common cause of fetal disease and abortion in Wyoming.     

Transposon (Tn5)-mediated suppressive integration of ColE1 derivatives into the chromosome of Escherichia coli K12 (dnaA)

While integration of ColE1 had not been observed previously by ordinary suppressive integration, a $\text{dnaA}$ (Ts) $\text{E. coli}$ strain with Tn$\text{5}$ at various sites of the chromosome and ColE1 or its mini-derivative, pAO3, but not pSC101, inserted by the same transposon produced integratively suppressed strains depending on the RecA function. In contrast to Hfr strains made with a stringently controlled plasmid, they contained the plasmid not only in an integrated but in an autonomous state at an amount comparable to the strain containing the plasmid only autonomously. Introduction of a RecA-deficient mutation to the strain with an integrated ColE1 derivative through conjugation failed. This is likely to be due to lethality of such a strain without RecA-dependent excision of the integrated high copy number plasmid or to quantitative deficiency of DNA polymerase I in addition to the $\text{recA}$ mutation.     

Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of cortisol and cortisone.

Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N⁴,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the $\text{in}\text{vitro}$ growth of L1210 by 50% (ED₅₀) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED₅₀ 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of $\text{T}\text{C}$ at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of $\text{T}\text{C}$.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.