Opposing effects of human peripheral blood lymphocytes on the growth of cultured leukemia cell lines

Natural killing of two human leukemia cell lines (K562 and Molt 4) in a soft-agar, clonagenic assay was shown to be the result of two opposite yet concurrent processes: target cell colony stimulation and inhibition. The stimulatory effect was demonstrable when the effector lymphocytes and target cells were separated in contiguous agar layers, suggesting mediation by a soluble factor. Similarly, stimulation occurred when the effector lymphocytes and target cells were combined at low effector-target cell ratios that do not favor direct cell contact. Target colony inhibition was found to be dominant when large E:T ratios were employed. Both target-effector binding and natural killing were significantly reduced in medium devoid of divalent cations.     

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Replication and amplification of recombinant plasmid molecules as extra chromosomal elements in transformed mammalian cells

Mouse thymidine kinase negative (TK^?) L cells, F4-12B2TK^? Friend erythroleukemic cells and hamster BHKTK^? cells were transformed in the absence of carrier DNA with closed circular molecules of herpes simplex virus 1 TK DNA cloned in plasmid pAT153 (pTK-1). The physical status of donor DNA in the transformed cells was studied by Southern blot hybridization and spot hybridization techniques. Up to 65 copies of pTK-1 DNA molecules/cell were present in transformed cells grown under selective conditions. Whereas a steady increase in the number of pTK-1 copies/cell was found during the first few weeks of growth in selective medium, 2–8 months later copy numbers varied within the same cell line and their numbers rarely exceeded fifty copies/cell. Donor DNA sequences were found both in the Hirt precipitate and in the supernatant showing that some of the pTK-1 molecules existed in circular form. Many of the cell lines gave a Southern blot hybridization pattern indicative of pTK-1 sequences integrated into high molecular weight DNA as well as present in a circular configuration.     

Differential diet breadths and species coexistence in leafroller-hunting eumenid wasps

There are, at least, three possible ways in which similar species coexist; resource partitioning, interference competition, and exploitation competition. Here, I investigated which way contributed to the coexistence of leafroller-hunting eumenid wasp species. Resource partitioning and, in addition, differential diet breadths proved to promote species coexistence in this case. First, I analyze the prey records and diet overlap of four eumenid species in a local area. The larger two eumenids hunted similar-sized prey items and had similar potential taxonomic prey uses. But the diet breadth of the subsocial eumenid was much wider than that of the solitary one. As a result, the diet overlap between the two large eumenids decreased. This was because the solitary eumenid attend repeatedly to the same hunting site inhabited by one abundant prey species, while the subsocial one made random hunting. On the other hand, the two medium-sized eumenids partitioned resources according to prey size. Secondly, I related these results to prey choice by several other species of eumenid obtained from literature sources. Ten Japanese common eumenids were divided into four groups according to their prey size. In each of the four groups, 2 to 3 wasp species differentiated the habitat (1 group) or coexisted by means of differential diet breadths (parallel with differential sociality, 2 groups).     

The genus Testudinella (Eurotatoria : Gnesiotrocha : Testudinellidae) in North-Eastern India

Nine species and subspecies of the genus Testudinella are reported from North-Eastern India. Of these, six are new to India and eight to the North-Eastern region. Remarks are made on their distribution.     

Chemical quantitation of hemoglobin glycosylation: fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ?-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia.This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.     

The effect of chlorpromazine on SCE frequency in human chromosomes: An in vitro and in vivo study

Schizophrenic patients who were receiving, or who had received chlorpromazine showed SCE levels similar to those in a normal control population. Of 8 normal individuals whose lymphocytes were exposed in vitro to chlorpromazine (0.05–2.00 μg/ml) for two cell cycles, 4 showed a significant increase in SCE, 3 showed no increase and 1 a decrease compared with untreated lymphocytes. Lymphocytes from a further 8 donors treated with 2.0 μg/ml chlorpromazine prior to mitogen stimulation (G₀ lymphocytes) showed a similar SCE response. Only 3 of the 8 donors showed a significant increase in SCEs over the baseline level. When proliferating lymphocytes were exposed to chlorpromazine 38 h after culture initiation and prior to the addition of BrdUrd to the culture medium, metaphase chromosomes from only 3 of the 8 individuals studied showed increased levels of exchange. These results indicate that chlorpromazine can induce SCEs in vitro but that there is considerable variation in SCE response among individuals. Furthermore, our data emphasises the importance of using more than 1 or 2 donors when analysing SCE response in human chromosomes.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Morphine addiction and withdrawal alters brain peptide concentrations

This study demonstrates that, during morphine addiction and withdrawal in rats profound alterations in the concentrations of a variety of brain peptides occur. Somatostatin, cholecystokinin, neurotensin and substance P concentrations increased during morphine addiction. Naloxone-induced withdrawal decreased brain concentrations of TRH, somatostatin, neurotensin and substance P. Naloxone alone decreased thalamic substance P and neurotensin concentrations. Vasoactive intestinal peptide concentrations were unaltered by any of the treatments. The fall in the tissue concentration of somatostatin during naloxone-induced withdrawal correlated well with the fall in the circulating growth hormone, suggesting that this could be secondary to somatostatin release. Our data support the hypothesis that brain peptides, acting locally in the brain as neuromodulators, play an important role in the genesis of the syndromes of morphine addiction and withdrawal.