A simple, rapid method to process and assay fatty acids and alcohols by gas chromatography

A rapid method is presented for the direct qualitative and quantitative assay by gas chromatography of microbial metabolites produced in culture and biological specimens. We have used this technique for analysis of metabolic end-products of anaerobic bacteria in broth cultures and in feces, but it would be suitable for studies with other organisms and biological specimens as well. Aqueous samples are injected directly into the system.Two gas chromatograph instruments are connected to one recorder, and employ thermal conductivity (TCD) and flame ionization detectors (FID). The column packings (Resoflex LAC-1-R-296 standard concentration (P)¹ for the TCD and 6% FFAP² on Porapak Q³ for the FID) permit separation of lower-chain volatile fatty acids (VFA's), alchohols and similar components as well as methyl ester derivatives of nonvolatile acids (lactic, pyruvic, and succinic) in a comparatively short time. Quantitation of the VFA's is provided by means of an electronic digital integrator.     

Effect of proteolytic inhibitors on growth and surface architecture of normal and transformed cells

Protease inhibitors were tested for their effect on the growth of normal and SV40-transformed mouse fibroblasts. The protease inhibitors TAME¹ and EWTI¹, which act competitively on proteases, reduce the growth of transformed cells more than that of untransformed parent cells. However, transformed cells grown in medium containing these drugs do not show contact inhibition of cell division or decreased agglutinability with Concanavalin A. The inhibition of growth is due to an extended duration of all phases of the cell cycle. The protease inhibitor TLCK¹, an active site titrant reacting irreversibly with trypsin, blocks transformed cells in the premitotic stage of the cell cycle. This effect does not occur in the untransformed parent cells. The decrease in agglutinability of transformed cells treated with TLCK is correlated with a partial synchronisation in the G2 stage of the cell cycle. Our results do not support the hypothesis that protease inhibitors induce transformed cells to assume a normal growth pattern and that this is accompanied by a decreased agglutinability with plant lectins.     

Purification of a serum DNA binding protein (64DP) with a molecular weight of 64,000 and its diagnostic significance in malignant diseases

A DNA binding protein with a molecular weight of 64,000(64DP) has been purified to homogeneity from human serum, and its quantitative assay has been developed. The average level of serum 64DP in 30 normal controls was 41.4 μg/ml, whereas it was 175 μg/ml in 87 patients with untreated malignant disease. Furthermore it was found to be elevated in all tested patients, 8 cases, with carcinoma in early stages. Serum 64DP has been found to be different from C3DP, CEA^# or α-FP^#, and it appears that this protein might prove to be a useful tumor marker in malignant diseases.     

Prolonged spring oestrus in mares: The use of progestogens with specific reference to proligestone

The problems of prolonged spring oestrus in mares, which is often associated with multifollicular ovaries (MFO), are described in the context of the need to produce foals early in the year. The results obtained, in terms of pregnancy and time of ovulation, after the intramuscular administration of a single standard 15-ml (1500 mg) dose of proligestone^a are described and compared with data available for allyl trenbolone^b, which is given orally, and with accepted data for non-medicated mares. It is concluded that proligestone may be useful in treating MFO and that its use may even aid conception. This latter observation is worthy of further investigation.     

Vinblastine potentiates the secretagogue action of dibutyryl cyclic AMP on the exocrine pancreas

The effects of VB⁴ on both structure and function of the pancreatic acinar cell have been examined in vitro. VB potentiates the secretagogue effect of DbcAMP⁵ but fails to affect the spontaneous release of enzymes. This potentiation is parallel to the disappearance of cellular microtubules and depends on the dose and the time of exposure to VB. The potentiation is energy dependent; it is more obvious in calcium containing medium than in absence of calcium. A damaging effect of VB an acinar cells is ruled out. These findings suggest the participation of microtubules in the secretory cycle of the pancreatic acinar cell.     

Contractions of rabbit testes in vitro: Permissive role of prostaglandins for the actions of calcium and some smooth-muscle stimulating agents

Rinsing actively contracting rabbit testes $\text{in vitro}$ with fresh Tyrode's solution abolished capsular contractions and the response of this preparation to either Ca⁺⁺, serotonin, or acetylcholine. Adding exogenous prostaglandin E₂ (PGE₂) to the medium restored contractility and the responses of the preparation to each of the above three agents.⁴ A reciprocal dependency was observed between Ca⁺⁺ and PGE₂ in stimulating contractions. PGE₂ potentiated, but was not required for the stimulatory action of either epinephrine or histamine. The stimulation of contractility by epinephrine, but not prostaglandin was inhibited by the α-blocking agent, ergotamine tartrate.⁴ This action of epinephrine did not involve prostaglandin release, nor was it inhibited by indomethacin pretreatment.⁴ Isoproterenol inhibited testicular contractions evoked by PGE₂.     

Multiple fraction collection using a peristaltic pump and a fraction collector modified for multiple channel operation

The fractionation of gradients in sedimentation analysis of proteins is a time-consuming operation. This operation can be performed more rapidly by using a multichannel peristaltic pump and two or more fraction collectors. Specifically designed fraction collectors are available for multiple fraction collection, notably the Slave Micro-Fractionator (Model SFC-80)¹ from Gilson Medical Electronics, Inc. Use of multiple fraction collectors during the fractionation of gradients has the disadvantage that these units occupy considerable space and are expensive. In addition, the total number of fractions collected from one gradient is often considerably less than the capacity of most fraction collectors. To offset the space and expense disadvantages, we have devised a modification for the Gilson Micro-Fractionator (Model FC-80H)¹ which permits three sets of 25 fractions each to be collected simultaneously from 3 gradients using only 1 fraction collector. A multichannel peristaltic pump is employed, and an attachment which permits three drop tubes to be positioned above the rack of fraction tubes is secured to the drop detector head¹ of the Micro-Fractionator. One drop tube is clamped in the normal manner in the drop detector head, and fraction size is determined by counting drops in the normal manner. Fractions from the other two drop tubes are not counted. Alternatively, fraction size can be determined by adjusting the pump speed and using the timer on the fraction collector.     

Ovulation and fertility after PRID,PRID + GnRH and GnRH in anestrous buffaloes

Two experiments, one in the winter (January) and the other in the summer (June), were undertaken to treat anestrous buffaloes at a farm. During experiment I, animals were treated with a progesterone releasing intra-vaginal device (PRID)^a, whereas during experiment II, animals were allocated to PRID + GnRH^b (125 μg) and GnRH-treated (250 μg). In both the experiments, animals were retained as untreated controls. All the treated animals were clinically examined four days post-treatment and inseminated two or three times at 24-hour intervals. The ovulation and fertility rates of PRID, PRID + GnRH and GnRH-treated animals were 25 and 12 %, 43 and 14 %, and 11 and 0 %, respectively. None of the controls exhibited estrus during the course of the study.     

Actin mediated calcium dependency of actomyosin in a myxomycete

A fraction obtained from $\text{Physarum polycephalum}$ by differential centrifugation displays magnesium adenosine triphosphatase activity; at low ionic strength (0.07 M KCl) the rate at which ATP¹ is split in 0.1 mM CaCl₂ is from 1.5 to 6.6 times the rate in 1 mM EGTA¹. Both actin and myosin are present in this fraction. On SDS gels several polypeptide bands are present in the range of 39,000 daltons to 14,000 daltons as well as those of actin and myosin. The addition of desensitized rabbit muscle actin to the fraction increased the rate of ATP splitting in EGTA, thereby decreasing the EGTA inhibition 30–50%. We conclude that actomyosin regulation by calcium in this acellular slime mould is, at least in large part, mediated through actin.     

The influence of cholesterol on the activity of phospholipids in blood coagulation: requirement for a liquid-crystalline lipid phase

A series of blood clotting tests including a specific activated factor X assay⁷, a thromboplastin generation test (TGT)² and a partial thromboplastin time (PTT) have been used to examine the activity of phosphatidyl serine (PS) in the presence and absence of cholesterol. In all the tests, PS from bovine brains showed a high level of activity which was potentiated by cholesterol to varying degrees. Cholesterol alone had no effect. Hydrogenated PS alone showed no activity but in all cases activity was largely restored by addition of cholesterol. The restoration of activity was shown to be related to the transition temperatures of the lipid mixtures in water as measured by differential thermal analysis. It was concluded that a liquid-crystalline phase is essential for the activities observed. Cholesterol may also act as an inert spacer for the acidic phospholipid molecules.     

Activation of the NADH-methemoglobin reductase reaction by inositol hexaphosphate.

Inositol hexaphosphate (IHP)¹ increases the rate of the NADH-methemoglobin reductase reaction at neutral pH. This effect is apparently associated with the change in conformation of methemoglobin induced by its interaction with IHP. It is consistent with the suggestion that IHP stabilizes the quarternary T type of structure in methemoglobin.     

Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase

The hepatotoxin thiobenzamide is S-oxidized by the microsomal flavin-containing monooxygenase (MFMO)¹ in liver, lung, and kidney of rabbit, mouse and rat. Its oxidation is accompanied by a large spectral shift which can be used as the basis of a simple convenient photometric assay for the MFMO system.     

Alcaloïdes de Zanthoxylum decaryi: la decarine,nouvel alcaloide dérivé de la benzophénanthridine

Four alkaloids have been isolated from the stem bark of Zatithoxylum decaryi. Three are known, dictamnine skimmianine, 4-methoxy-1- methyl-2-quinolone. The fourth decarine is new; its structure has been established as 9-methoxy-10-hydroxy-2,3-methylenedioxybenzophenanthridine.¹     

Arylazido aminopropionyl ATP, and active site directed photoaffinity reagent for mitochondrial adenosine triphosphatase.

The photoaffinity label, arylazido aminopropionyl ATP¹, brings about a photodependent inhibition of mitochondrial F₁-ATPase and an associated specific covalent labeling of the soluble enzyme. In addition the ATP analog can act as a substrate when incubated with F₁-ATPase in the dark.     

Solubilization from skeletal muscle of two components that specifically bind -bungarotoxin

BuTX⁷ binds irreversibly to two components of skeletal muscle, one with apparent molecular weight (by gel filtration) of 550,000 and one of 200,000. Only the former, thought to contain the acetylcholine receptor, is elevated in denervated muscle, about 20-fold at its maximum. After solubilization, these components retain their specific properties.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.     

Reactivation of progessive motility in hamster sperm modified by triton X-100

Epididymal hamster sperm, denuded of their plasma membrane with Triton X-100 and washed free of cytosol, were found to become progressively motile in defined media containing ATP¹ and salts. The chemical requirements for motility were investigated and several experimental observations were made that were relevant to the mechanism and control of sperm motility.     

Phencyclidine stimulates guanylate cyclase activity.

The hallucinogenic agents, phencylidine (Angel's Dust), TCP¹ and their morpholine analogs enhanced the activity of guanylate cyclase {E.C.4.6.1.2}, the enzyme that catalyzes the production of guanosine 3′, 5′-monophosphate. This activation of guanylate cyclase by hencyclidine and TCP was observed over the concentration range of .00001 mM to 1 mM, while the morpholine analogs stimulated tha activity of guanylate cyclase in concentration of .0001 mM to 1 mM.     

Formylation of enzyme-bound valine and stepwise elongation of intermediate peptides of gramicidin A by a cell-free enzyme system.

A protein, tentatively named apo-Q-protein I, with molecular weight of 15,000¹ from the reconstitutively active cytochrome $\text{b-c}{}_1$ complex has been identified as being responsible for electron transfer between succinate dehydrogenase and ubiquinone. The identification was based on the chemical modification, proteolytic enzyme digestion, and isolation and purification of the protein to nearly pure form.