Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P-nmr spectra of phospholipid vesicular membranes and their temperature variation

1. Shifts in the ¹H and ³¹P-nmr signals originating from the outer and inner phosphorylcholine head-groups and from the lipid acyl chains are observed when phosphatidylcholine vesicles are treated with increasing extravesicular concentrations of the lanthanides Eu³⁺, Pr³⁺, Yb³⁺, and Dy³⁺. 2. The addition of KNCS to increase the binding of the lanthanide ions to the outer head-groups is used to demonstrate that the intravesicular group shifts are not caused by bulk susceptibility effects. 3. The magnitude and direction of the observed shifts in the ¹H-nmr spectrum are shown to be consistent with (a) pseudocontact interaction of the paramagnetic lanthanide ions with the outer phospholipid head-groups, (b) current views of the conformation of the phosphatidylcholine head-group in the presence of lanthanides, and (c) a conservation of magnetic field within the vesicles due to their spherical nature. 4. Variation of the shifts with temperature are compared for egg phosphatidylcholine and dipalmitoyl phosphatidylcholine. The temperature variation in shifts is also used to study phase transitions in each monolayer and phase separations in mixed lipid systems.     

What is “language” that can facilitate the flow of information? a contribution to a fundamental theory of language and communication

A theory of language is derived for complex systems by contrasting the difference between “hard” systems and “soft” systems. Complexity in a field system made up of atomistic entities is defined by the capability of the atomistic-like entities in soft systems to absorb energy internally into fluid-like, gel-like, dissipative processes rather than to equipartition energy rapidly among all degrees of atomistic freedom. Language then emerges as the “mechanistic” linkages that can catalytically switch or evoke changed atomistic states in such “soft” systems. A note on the source of syntax is also provided.     

Relationship of In Vivo MR Parameters to Histopathological and Molecular Characteristics of Newly Diagnosed,Nonenhancing Lower-Grade Gliomas

The goal of this research was to elucidate the relationship between WHO 2016 molecular classifications of newly diagnosed, nonenhancing lower grade gliomas (LrGG), tissue sample histopathology, and magnetic resonance (MR) parameters derived from diffusion, perfusion, and ¹H spectroscopic imaging from the tissue sample locations and the entire tumor. A total of 135 patients were scanned prior to initial surgery, with tumor cellularity scores obtained from 88 image-guided tissue samples. MR parameters were obtained from corresponding sample locations, and histograms of normalized MR parameters within the T2 fluid-attenuated inversion recovery lesion were analyzed in order to evaluate differences between subgroups. For tissue samples, higher tumor scores were related to increased normalized apparent diffusion coefficient (nADC), lower fractional anisotropy (nFA), lower cerebral blood volume (nCBV), higher choline (nCho), and lower N-acetylaspartate (nNAA). Within the T2 lesion, higher tumor grade was associated with higher nADC, lower nFA, and higher Cho to NAA index. Pathological analysis confirmed that diffusion and metabolic parameters increased and perfusion decreased with tumor cellularity. This information can be used to select targets for tissue sampling and to aid in making decisions about treating residual disease.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

alpha 1-Adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin.

Binding of (³H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (³H)-prazosin bound/mg protein and the K_D was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (³H)-prazosin as follows: (?)adrenaline > (?)noradrenaline > (?)phenylephrine ? ($\text{+}$)isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ? piperoxan > yohimbine > sulpiride > propranolol. The K_D for beta-adrenoceptors assessed by (?³H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (³H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha₁) adrenoceptors in myocardial tissue.     

Alteration in gene expression at the onset of human Y-79 retinoblastoma cell differentiation

We have tested the hypothesis that differentiation and growth arrest of Y-79 human retinoblastoma cells in culture is associated with a modification of gene expression. We first examined proteins translated from mRNAs isolated from Y-79 cells growing in suspension and in attachment cultures in serum-containing medium and found them to be markedly different. This suggests that membrane-substrate interactions are of major consequence in the biochemical differentiation of these cells. Secondly, we examined the patterns of proteins translated from attached cells which had been induced to morphologically differentiate into neuronal-like and glial-like cells by serum-withdrawal and dibutyryl cAMP treatment respectively. The in vitro translatable proteins of mRNAs isolated from these cultures were found to be markedly different from those of the suspension and attachment cultures. Thirdly, we found that treatment of cells growing in attachment culture in serum-containing medium supplemented with 8-bromo cAMP, butyrate and retinoic acid as well as dibutyryl cAMP resulted in discreet alterations in proteins translated in vitro from extracted mRNAs. Although all these substances inhibit the growth of Y-79 cells, only dibutyryl cAMP and butyrate result in morphological differentiation of cells. Our results suggest that (1) attachment and morphological differentiation of Y-79 cells are both related to specific alterations in gene expression and (2) differentiation and inhibition of cell growth by various agents can be correlated with changes in translatable mRNA species although all agents do not act in the same mode.     

Membrane potential and surface charge densities as possible generalized regulators of membrane protein activities

On the basis of experimental evidence accumulated in studies of coupling and other membranes a theoretical discussion of a “breathing” membrane structure dependent upon a changing transmembrane electrical (electrochemical) gradient is presented. In the case of certain membranes difference of electrical potentials across the membrane (Δψ) is considered as one of important driving forces for some membrane protein folding and/or the arrangement of subunits within some oligomeric proteins. Δψ changes may induce synchronous reversible energetically favourable transitions in the three-dimensional architecture of several spatially separated proteins of the biomembrane. A polyfunctional regulation of levels of activities of spatially separated membrane proteins may be achieved; in some cases alterations of Δψ and surface charge densities may fulfil function of a link in the chain of events triggering membrane-bound activities in response to external stimuli.     

Symbolic logic analysis of cause of dealth in humans: Application to 108 patients after coronary artery bypass graft surgery

Over the past two decades there has been increasing interest in the development of an objective, or formalized “medical logic”, and many authors have employed classical symbolic logic as a part of their approach. On the other hand, it has become clear that certain patterns of reasoning which are commonplace in evaluating patients clinicopathologically are awkward to handle in classical symbolic logic. The present paper proposes an extension of classical symbolic logic which addresses three problems in medical reasoning: (i) the problem of provisional diagnosis, (ii) the problem of inaccessible data, and (iii) the problem of the adequate discharge summary. It is proved mathematically that with a suitably constructed logic, the system “complains” until all questions involving threats to the patient's health are either answered or shown to be unanswerable because of inaccessibility of data. To illustrate this method, the cause of death was studied in 108 patients who had been autopsied at The Johns Hopkins Hospital after coronary artery bypass surgery. The analysis disclosed that 46% of patients suffered a fatal complication which could be attributed to events in the perioperative period; in 15% of patients the cause of death was unexplained by the analysis. Computerized symbolic logic analysis is a useful supplement to intuitive reasoning in assigning cause of death to patients with complex medical histories.     

Radiorespirometry and metabolism of cultured cells attached to petri dishes

A radiorespirometer was constructed for continuous quantitation of ¹⁴CO₂ released from specifically labeled substrates by intact cultured cells attached to plastic petri dishes. An airtight chamber is created by sealing the petri dish with a specially designed cover inside a thermostated holder. Rapid equilibration of released ¹⁴CO₂ with a 5% $\text{CO}{}_2\text{95\%}$ air carrier gas is achieved by bubbling the carrier gas under the surface of the growth medium. Labeled CO₂ is removed from the carrier gas by trapping in an organic base and quantitated by liquid scintillation counting. Additions to or sampling of the growth medium may be performed during a run and the carrier gas may be modified to test the effects of anesthetics and different O₂ levels. The ability to continuously monitor ¹⁴CO₂ release can provide valuable information concerning the metabolic pathways of substrate oxidation which cannot be obtained from single ¹⁴CO₂ determinations. A capacity of 12 culture plates enormously increases the amount of data that can be collected in a given time. The use of liquid scintillation counting increases the sensitivity and resolution over the ion chamber and Geiger counter methods, and permits utilization of the procedure in a much wider range of laboratories. Data obtained for the oxidation of specifically ¹⁴C-labeled glucose and pyruvate by neonatal rat heart cells in culture, in both the presence and absence of oxygen, are provided as examples of the utility of the method.     

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E–BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR = 0.49; 95% CI, 0.25–0.95; P = .036) and in tumor cells (HR = 0.37; 95% CI, 0.18–0.79; P = .010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.     

In vitro stimulation of cell death in the moulting glands of Oncopeltus fasciatus by 20-hydroxyecdysone

The moulting glands of the milkweed bug, Oncopeltus fasciatus, normally degenerate just before the time of ecdysis to an adult (day 7 of the fifth instar). Morphologically normal cell death can be prematurely stimulated in vitro by 20-hydroxyecdysone. Breakdown is triggered by a 24-hr period of exposure to 20-hydroxyecdysone, but an additional incubation period is required before clear signs of degeneration are manifested. Glands removed after the onset of endogenous ecdysteroid secretion degenerate in vitro in the absence of added hormones. Thus, in the moulting glands of Oncopeltus, ecdysteroids appear to act as an important trigger for metamorphic cell death.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.     

Surfactant ion-pair high-performance liquid chromatography of tryptophan and some of its metabolites in biological fluids

A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.     

Isolation of a non-thionein copper-binding protein from liver of copper-injected rats

1. The characterization of a low molecular weight, non-thionein, Cubinding protein isolated from rat liver is reported. The protein was isolated following chronic administration of Cu(NO₃)₂ using a combination of Sephadex G-75 and Sephadex DEAE A-25 chromatography. The protein did not bind to fully equilibrated Sephadex DEAE which formed the basis of the isolation procedure. 2. The final protein pereparation was found to be homogeneous by a variety of electrophoretic techniques and was distinguished from metallothionein on the basis of its behaviour on ion exchange and electrophoretic systems, spectral properties, and amino acid composition and metal content. It contains 6.8% cysteine and was found to bind Cu in a ratio of 1.5:1 based on a molecular weight of 11 000. 3. These results confirm the necessity to use techniques other than gel filtration alone to obtain adequate separation of low molecular weight metal-binding protein fractions.