Preparation and biological activity of taxol acetates

Synthesis and biological activity of 2′-acetyltaxol and 7-acetyltaxol are reported. Activity is measured $\text{in}\text{vivo}$ by cytotoxicity toward the macrophage-like cell line J774.2, and $\text{in}\text{vitro}$ by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2′ results in loss of $\text{in}\text{vitro}$ activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on $\text{in}\text{vitro}$ microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for $\text{in}\text{vitro}$ activity and this position is available for structural modifications.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

The biological activity of some siderochrome derivatives

Semisynthetic derivatives of natural trihydroxamic acid iron chelates, or siderochromes, have been prepared and tested for biological activity. The activity of these compounds as growth factors for $\text{Arthrobacter}$$\text{JG-9}$ or as antagonists to the siderochrome antibiotic, albomycin, is relatively unaffected by structural alteration. However, their ability to function as ferric ionophores for the fungus, $\text{Ustilago}$$\text{sphaerogena}$, is very sensitive to minor structural variations. Branching at the β-position of the hydroxamate acyl function leads to loss of all biological activity.     

Stabilization of proteins immobilized on Sepharose from leakage by glutaraldehyde crosslinking

A technique has been developed to prevent leakage of proteins immobilized on Sepharose without destroying their biological functions. This involves the use of glutaraldehyde at concentrations ranging from 0.015 to 0.25% ($\text{v}\text{v}$) to crosslink proteins, which had been coupled to Sepharose by conventional methods. Glutaraldehyde crosslinking decreases immuno-globulin G leakage from Sepharose-immunoadsorbents to undetectable levels without noticeably affecting antigen-binding activity and reduces leakage of lactoperoxidase from solid-phase lactoperoxidase with only a moderate reduction of enzymatic activity.     

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH₂S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the $\text{in}\text{vitro}$ activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro¹, D-Phe², D-Trp^3,6]LH-RH antagonist was completely inhibitory at 30 ng $\text{in}\text{vitro}$ and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed $\text{in}\text{vivo}$ antiovulatory activity at the 250 μg level.     

Alkaline isomerization of ferricytochrome C from Euglena gracilis

$\text{Euglena gracilis}$ ferricytochrome $\text{c}$ has a small absorption maximum at about 700 nm having an extinction of 850 ± 10 M^?1cm^?1. This absorption band is analogous to the more commonly found maximum at 695 nm which is observed in ferricytochromes from other sources and which is characteristic of ligation of methionine 80 with the heme ion. The 700 nm band disappears upon raising the pH to 11 giving a transition involving a single proton having an apparent pK of about 10. These results demonstrate that the phenolic ionization of tyrosine 67 is not required to trigger the alkaline isomerization of ferricytochromes $\text{c}$ since Euglena cytochrome has a phenylalanine residue at position 67.     

Procedure for measurement of amino acid transport from blood to brain in small animals

The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [¹⁴C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity $\text{S}$ and the measured specific activity S in any individual animal. $\text{S}$ is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by $\text{S}$ gave the tissue Tyr uptake U. The function $\text{dU}\text{dt}$ is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l-methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.     

Myelin basic protein: a substance that releases immunoreactive growth hormone in vitro.

A protein has been isolated from ovine hypothalamus on the basis of its ability to stimulate release of growth hormone by $\text{in}$$\text{vitro}$ cultures of dispersed pituitary cells. This protein has been identified as being myelin basic protein. With no similar biological activity $\text{in}$$\text{vivo}$, myelin basic protein is thus to be recognized as a potentially interfering substance in any search for the physiological growth hormone releasing factor using $\text{in}$$\text{vitro}$ assay systems.     

Role of histidine residues in ovine lutropin: effects on steroidogenic activity.

The modification of histidine residues of ovine pituitary lutropin by rose bengal sensitized photooxidation has been investigated. The destruction of an average of one histidine out of six lead to 90% loss of biological activity as examined by the $\text{in}\text{vitro}$ steroidogenic response in the rat Leydig cell essay. Further modification of an average 2 – 3 histidine residues reduced the biological activity to less than 1% of the native lutropin. The modified lutropin was incapable of inhibiting the native lutropin induced steroidogenesis. Gel filtration experiments and polyacrylamide disc gel electrophoresis patterns indicated that no dissociation of the molecule into subunits occurred. This is the first report on the essentiality of the histidine residue for the activity of lutropin.     

In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of tyrosine hydroxylase

Studies are presented which demonstrate that rat pheochromocytoma tumors are a convenient material for the preparation of tyrosine hydroxylase mRNA. Total pheochromocytoma poly(A)⁺mRNA has been extracted from tumors, then translated in a reticulocyte lysate cell-free system. Neo-synthesized tyrosine hydroxylase has been identified by direct immunoprecipitation followed by sodium dodecyl sulfate acrylamide gel electrophoresis. The proportion of this specific mRNA has been calculated; it represents 0.15 per cent of the total poly(A)⁺mRNA. The molecular weight of the $\text{in}\text{vitro}$ synthesized tyrosine hydroxylase is 62,000.     

The artifactual nature of fluoride inhibition of reverse transcriptase and associated ribonuclease H

Surprisingly, the $\text{sn}$-1 configuration of 1-0-hexadecyl-2-acetyl-glycerylphosphorylcholine showed significant activity, 3.22 × 10^?9 M, when compared to the $\text{sn}$-3 enantiomer, 2.92 × 10^?10 M and a racemic mixture with a value of 7.2 × 10^?10 M. A methoxy substitution at the C-1 or C-2 position of octadecyl glycerylphosphorylcholine gave a derivative with high biological activity for stimulating serotonin release from rabbit platelets. A 1-0-dodecyl-2-methoxy analogue showed very low activity; also, a comparable series of 0-benzyl derivatives were inactive. Examination of 1-0-hexadecyl, 1-0-octadecyl- or 1-0-dodecyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine showed that the hexadecyl compound had three times the biological activity of the octadecyl and five times that of the dodecyl.     

3-Epi-20-hydroxyecdysone from meconium of the tobacco hornworm

A new ecdysteroid, 3-epi-20-hydroxyecdysone, with biological activity $\text{1}\text{10}$ to $\text{1}\text{15}$ that of α-ecdysone has been isolated and identified from the meconium of the tobacco hornworm. The possible role or function of this steroid as an inactivation product and/or regulator of molting hormone titer is discussed.     

Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

The interaction of chloride ions with human hemoglobin

The immobilization of glucose oxidase, a glycoenzyme from $\text{Aspergillus}$$\text{niger}$ consisting of 16% carbohydrate, has been achieved by oxidizing its carbohydrate residues with periodic acid followed by coupling the activated enzyme to water-insoluble $\text{p}$-aminostyrene. At pH 5.6 and 25°, approximately 60% of the carbohydrate residues are oxidized, but the enzyme retains full activity. No oxidation of any amino acid residue is evident. The enzyme-polymer conjugate derived from this activated enzyme retains full activity and even shows a slightly enhanced thermal stability at 60° compared with the soluble native and oxidized glucose oxidases.     

Inhibition effect of rosette formation between human lymphocytes and sheep erythrocytes by specific heptapeptide isolated from uremic fluid and its analogs.

One of middle molecular substances (H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH) and its two analogs, in which the proline residue in position 2 was replaced by glycine and the alanine residue in position 3 was replaced by valine exert a inhibition effect on $\text{in vitro}$ E-rosette formation. Its synthetic two analogs showed diminished biological activity compared to native heptapeptide.     

Tuftsin: A hormone-like tetrapeptide with antimicrobial and antitumor activities

A specific fraction of immunoglobulin G binds to polymorphonuclear neutrophils and stimulates their phagocytic activity. This phagocytosis-stimulating activity resides solely in a small peptide termed tuftsin, of the sequence Thr-Lys-Pro-Arg, which has been isolated from the leukophilic immunoglobulin G fraction. The physiological significance of tuftsin has been demonstrated in splenectomized patients and patients with a congenital tuftsin abnormality, in whom the low levels of tuftsin in sera (measurable by radioimmunoassay) coincides with a high incidence of infection. Tuftsin has also been shown to enhance bactericidal activity in addition to phagocytosis. Its biological activities appear to be mediated via specific tuftsin receptors which have been found on macrophages, monocytes and granulocytes. In addition, tuftsin possesses chemotactic, migration-enhancing and mitogenic properties for leukocytes and has recently been shown to enhance their anti-tumor activity $\text{in}$$\text{vitro}$ as well as $\text{in}$$\text{vivo}$. Other known activities of tuftsin include effects on the activity of the hexose monophosphate shunt, on the concentrations of intracellular cyclic nucleotides and on the efflux of Ca²⁺ in leukocytes. Tuftsin has been chemically synthesized in various laboratories using different procedures and also is available commercially. The above features of tuftsin plus the expected low toxicity of this peptide make tuftsin a very attractive agent for immunotherapy against infection and cancer. However, a great deal of caution needs to be exercised when using tuftsin due to inhibitory contaminants found in certain commercial preparations.     

Tyrosine's pressor effect in hypotensive rats is not mediated by tyramine

Tyrosine, the amino acid precursor of catecholamines, increases blood pressure (BP) in hemorrhaged hypotensive rats. Since tyrosine may also be decarboxylated to form $\text{tyramine}$^∞, which releases norepinephrine from sympathetic terminals, we tested the hypothesis that $\text{tyramine}$ formation might mediate tyrosine's ability to increase BP. Three lines of evidence indicate that tyrosine does not act via this mechanism: pretreatment with reserpine blocked $\text{tyramine's}$ but not tyrosine's pressor activity; pretreatment with hexamethonium left $\text{tyramine's}$ effect intact but blocked the pressor response to tyrosine; and plasma $\text{tyramine}$ did not increase after an hemodynamically-active dose of tyrosine (100 mg/kg).     

Structure activity relationship of lidocaine type local anesthetics.

The structure activity relationship of nine compounds were studied and compared to lidocaine. The extent of local anesthetic activity was ascertained by the tail pinch method in mice, and by the isolated frog sciatic nerve method. The effective and lethal dose in 50% of the animals was also determined. Three of the nine compounds appeared to possess significant local anesthetic activity in the $\text{in vivo}$ studies and thus were selected for further investigation $\text{in vitro}$. The $\text{in vivo}$ studies also indicated that two of the three were more toxic than lidocaine. The $\text{in vitro}$ results demonstrated that methyl substitution at positions 2,3 and 5 on the benzene ring produced a compound of slightly more anesthetic potency in an acid medium. At pH 7.8 all three compounds approached the potency of lidocaine. These data indicate that methyl substitution at other than the ortho position of the benzene ring generally results in compounds of lesser local anesthetic activity while tending to increase the toxicity.     

Characterization of 3-deoxy-D-manno-octulosonic acid as a component of the capsular polysaccharide antigen from Neisseria meningitidis serogroup 29-e

3-deoxy-$\text{D}$-$\text{manno}$ acid (KDO) has been characterised as the major component (53%) of the capsular polysaccharide antigen of $\text{N}\text{. meningitidis}$ serogroup 29-e. This is the first reported occurrence of KDO in any biological polymer other than its well established occurrence in the lipopolysaccharides of gram-negative bacteria.     

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.