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.     

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.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

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.     

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.     

Fractionation of chromosomal deoxyribonucleoproteins by partition in two-phase aqueous polymer systems

Deoxyribonucleoprotein (DNP)¹ prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately $\text{1}\text{3}$ of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio.     

Cobalt cytochrome c: enzymic assays.

An improved synthesis for cobalt-cytochrome $\text{c}$ has been developed; its half reduction potential is ?140 ± 20mV. Reduced ^Cocyt-$\text{c}$³ is oxidized by bovine heart cytochrome $\text{c}$ oxidase at a rate ～45% that of the native cytochrome $\text{c}$. It is not reduced by mitochondrial NADH or succinate cytochrome $\text{c}$ reductase nor by microsomal NADH or NADPH cytochrome $\text{c}$ reductase.     

A possible role for cytochrome P-450 during the biosynthesis of zymosterol from lanosterol by Saccharomyces cerevisiae

A cell-free system has been obtained from $\text{Saccharomyces cerevisiae}$ which is capable of efficiently converting lanosterol¹ to a mixture of 4-demethyl sterols, quantitatively the most important identifiable component of which was zymosterol. Little or no ergosterol was synthesized. In the presence of carbon monoxide, the rate of zymosterol biosynthesis from lanosterol was decreased by 57% compared with that observed in control incubations and the amount of unmetabolized lanosterol was greater. Mitochondrial electron transport inhibitors such as cyanide and antimycin A had no effect on the overall rate of 4-demethyl sterol biosynthesis from lanosterol nor on the degree of inhibition by carbon monoxide.     

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.     

Anomalous side chain amidation in plasma membrane proteins of simian virus 40-transformed lymphocytes indicated by isoelectric focussing and laser Raman spectroscopy.

Plasma membranes isolated from normal hamster lymphocytes and lymphoid cells transformed by SV40¹ have been compared. Isoelectric focussing in 1% Triton $\text{X-100}\text{8M}$ urea reveals higher isoelectric points than normal for the non-glycosylated proteins in the membranes of transformed cells. This suggests greater amidation of membrane aspartates and/or glutamates. The focussing patterns also reveal a shift to lower pH for the isoelectric points of most glycosylated proteins suggesting increased silalylation. The Amide I – Amide II laser Raman spectra of the two membrane categories are consistent with greater side chain amidation in the membranes of neoplastic lymphocytes.     

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.     

Protein phosphatase activity in acetylcholine receptor-enriched membranes.

Phosphorylation of the acetylcholine receptor (AChR)¹ has been demonstrated in AChR-enriched membranes prepared from the electric organ of $\text{Torpedo californica}$. In this report we show that the same AChR-enriched membrane fraction also contains phosphoprotein phosphatase activity which dephosphorylates both the endogenous AChR and exogenous phosphorylated casein. Release of [³²P] PO₄ from phosphorylated casein was shown to be inhibited by F^? as well as GTP. cAMP and cGMP were without effect. Dephosphorylation of the membrane-bound AChR was also inhibited by F^?. This phosphorylation-dephosphorylation mechanism may play a role in mediating the function of the AChR at the synapse.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.     

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.     

Generation of methionine and leucine-enkephalin from precursor molecules by cation-sensitive neutral endopeptidase of bovine pituitary

Highly purified preparations of cation-sensitive neutral endopeptidase, from bovine pituitary, and also rabbit brain, generate methionine-enkephalin, from α-endorphin, a peptide containing the amino acid sequence 61–76 of β-lipotropin (β-LPH), The enzyme also catalyzes the hydrolysis of the Leu-Thr bond in the synthetic peptide Tyr-Gly-Gly-Phe-Leu-Thr-2-naphthylamide with the release of leucine-enkephalin and Thr-2-naphthylamide. Neither Met- nor Leu-enkephalin are degraded. The data indicate that the presence of a free N-terminal group of tyrosine inhibits the further degradation of Leu- and Met-enkephalin by the endopeptidase. It is suggested that cation-sensitive neutral endopeptidase is one of the enzymes capable of generating Met- and Leu-enkephalin $\text{in}\text{,}\text{vivo}$.     

A mechanism for the hydrolysis of MgATP by actomyosin of skeletal muscle.

Based on a model of the active site of myosin (Ramirez, Shukla &; Levy, 1978), a chemical mechanism for MgATPase and intermediate oxygen exchange is presented. In this mechanism, oxygen exchange takes place via an oxyphosphorane intermediate that undergoes double turnstile rotation (Ugi, Ramirez, Marquarding, Klusacek &; Gillespie, 1971; Ramirez &; Ugi, 1974. During hydrolysis by native skeletal muscle myosin, only three [¹⁸O] atoms from labelled water are rapidly incorporated into the phosphorus that is finally released to the medium as P_i; whereas, during hydrolysis by subfragment 1 (S1), which is the head of myosin, four oxygens are labelled rapidly. To explain this difference, we postulate that cleavage of the (S1)-(S2) hinge in the preparation of S1 modifies the interaction of the oxyphosphorane intermediate at the active site. This enables a normally non-exchangeable oxygen to enter the exchange process. This is consistent with our earlier interpretation to the effect that the active site and the hinge in myosin are relatively close to each other Shukla &; Levy, 1977b; Shukla &; Levy, 1978. We postulate that the major elements of the active site are situated on a 92 amino acid fragment, p₁₀, isolated by Elzinga &; Collins, 1977 from myosin. P₁₀ is now known to be situated in the region that connects the head to the body of a myosin heavy chain (Lu, Sosinki, Balint &; Streter, 1978). An examination of the p₁₀ fragment for a possible point of proteolytic attack in the region of the hinge which will generate S1 revealed lysine 82. Breaking the protein chain at a point so close to the active site pocket could explain the effect of hinge cleavage on oxygen exchange. Two additional features of the present mechanism are: (1) the protonation of P_γ of a MgP_α,P_γ complex of ATP, which depresses monomeric metaphosphate mediated hydrolysis, and enhances oxyphosphorane formation by addition of water to P_γ; (2) the coordination of N^τ-methylhistidinet² of actin with Mg at the active site, which activates the release of the products of hydrolysis.     

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.     

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.     

Synthesis of d1-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxaprostaglandins oxaprostaglandins of the E₁ and F₁α series⁷ from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both $\text{in vitro}$ and $\text{in vivo}$ of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxa-PGE₁, methyl ester (VIII) are presented.