Hexokinase isozyme variability in Drosophila robusta

Several isozymes of hexokinase have been found in Drosophila robusta. These have been defined in this paper in several dimensions, including genetic variation, ontogenic variation, tissue-organ variation, and substrate specificity.The work reported in this paper was supported in part by USPHS grant AM 09381, USPHS Career Development Award 1-K3-AM 7959 (GJB), and by Contract AT (11-1)-1152, Atomic Energy Commission, and in part by the Research and Development Command, Office of the Surgeon General, Department of the Army, under Contract DA-49-193-MD-2855 with the Department of Medicine, University of Michigan. This is contribution No. 662 from the Army Research Program on Malaria.     

1,25-Dihydroxy vitamin D2 induces leukemia cell differentiation

This work supported in part by the NIH (USPHS), Council for Tobacco Research, American Institute for Cancer Research, and the Cornell Consolidated and Cornell Biotechnology Program which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the US Army Research Office and the National Science Foundation.     

The fine structure of the compound eye of a damsel-fly

Summary The compound eyes of two species of damsel-flies, Ishunura senegalensis and Cersion calamorum, were examined by electron microscopy. Each ommatidium is composed of eight retinula cells which are semistratified in the receptor layer. The retinula cells are divided into four types from the difference of levels in the rhabdom formation; one distal large cell having the rhabdomere only in the distal layer, four middle cells forming the rhabdom in the middle layer, two proximal cells making up the rhabdom in the proximal layer and one distal small cell having no rhabdomere in any layers. In addition, the lamina ganglionaris was partly observed. Some retinula axons terminate at an different level from the other axons. The functional differentiation among these different types of cells is discussed with relation to the analysis of the polarized light and the discrimination of the diffraction images.This work is supported by a grant from the U.S. Army Research and Development Group (Far East), Department of the Army (DA-CRD-AG-S29-544-67-G61).The authors wish to express their gratitude to Drs. H. Morita and H. Tateda for their helpful discussions throughout this study.     

The suppressor of forked mutation in Drosophila melanogaster: Interactions with the lozenge gene

An interaction between the lozenge gene and the suppressor of forked gene of Drosophila melanogaster has been investigated both spectrophotometrically and electrophoretically. The nature of this interaction is such that certain lozenge alleles appear to be phenotypically suppressed while others are enhanced or unaffected, and the results reported demonstrate that the effect can clearly be observed at the biochemical level. Earlier observations have suggested that the suppressor of forked gene codes for a ribosomal protein, and this hypothesis is discussed.These studies were supported by USPHS Grants GM-18485 and GM-20361 to P. D. S. P. D. S. is a recipient of USPHS Research Career Development Award GM-70758.     

Wartime research on malaria chemotherapy

Malaria was a major problem for the opposing forces in World War II. During the first year of operations in the South West Pacific the casualties caused by this disease greatly exceeded the numbers of battle casualties. In response to this situation comprehensive research and development programs to discover new antimalarial drugs were undertaken in the United States and Britain. In both countries compounds synthesised by co-operating chemical laboratories were screened against bird malaria and those with high activity and low toxicity were tested in man. The wartime program in America was funded by the Office of Scientific Research and Development and co-ordinated through a specially designated body under the Committee on Medical Research of the National Research Council. It was an enormous undertaking involving a massive co-operative effort between pharmacologists, chemists, and clinical research scientists from American universities, the US Public Health Service, and the laboratories of commercial pharmaceutical companies. The British program, on a much smaller scale, was based on a co-operative arrangement between the research laboratories of Imperial Chemical Industries at Manchester, the Liverpool School of Tropical Medicine and the British Medical Research Council. The wartime programs in both countries identified a number of promising leads but lacked the resources to permit their rapid clinical evaluation against field strains of human malaria. This deficiency was overcome by experiments conducted by the Land Headquarters Medical Research Unit of the Australian Army in Cairns, Queensland with the use of army volunteers. Large scale clinical trials of the most promising compounds which emerged from the American and British programs were carried out in Australia. This co-operative endeavour among allied scientists resulted in a range of new drugs which have had an enduring influence on malaria chemotherapy.     

Isolation and enrichment of the gastric chief cells of the rat

Summary The isolation and enrichment of the gastric chief cells of the rat are described. Ultrastructural examination showed 85% enrichment from a mixed population of mucosal cells following their centrifugation through a discontinuous Percoll gradient. When compared to homogenates of the initial mixed cell population, the enriched chief cell population showed over a three-fold increase in pepsin(ogen) content. Preliminary experiments showed that a combination of the secretagogues histamine and carbamylcholine caused a significant increase in pepsin release from enriched chief cell preparations and a concomitant decrease in their pepsin content as compared to untreated cells. The results obtained in this study indicate the feasibility of employing this procedure for the isolation of gastric chief cells for the in vitro study of secretagogue regulation of pepsin secretion.This work was supported by USPHS Grant # 20431. Dr. Rosenfeld's work is supported, in part, by Research Career Development Award # AM 00456 from the National Institute of Health     

Ultrastructural studies of vagal paraganglia in Syrian hamsters

Summary Typical vagal paraganglia of Syrian hamsters are encapsulated in connective tissue and consist of groups of epithelial cells. Ganglion cells, a few fenestrated capillaries, and bundles of unmyelinated nerve fibers are intermingled among the parenchymal cells. The parenchymal cells are of two types: chief or paraganglion and sustentacular or supporting cells. The processes of the supporting cells partly or completely surround the paraganglion cells. In addition to the nucleus, Golgi complex, mitochondria, parallel-arrayed granular endoplasmic reticulum, and lipofuscin pigment, the chief cells are characterized by the presence of numerous membrane-bound, electron opaque granules. After an injection of ³H-dopa, labelings were concentrated over the chief cells and were associated predominantly with the granules. Following glutaraldehyde-dichromate treatment the granules gave a positive reaction for unsubstituted amines. These results suggest that the chief cells contain catecholamines in the electron opaque granules.Research supported by USPHS Grants NS 05665, 00690 and HE 12751. A preliminary report of this research was presented before the American Society for Cell Biology, 1969.Sponsored by National Council on Science Development, Republic of China.Recipient of Career Research Development Award 1 K3 GM 28064.     

Quantitative studies of amino acid and growth factor requirements of transformed and nontransformed cells in high concentrations of serum of lymph

Summary The growth rate of spontaneously transformed BALB/3T3 cells is proportional to glutamine concentration between 50 and 400 μM, with little or no growth occurring in less than 50 μM glutamine. By contrast, nontransformed BALB/3T3 cells multiply, although slowly, with as little as 20 μM glutamine. Neither cell type depletes the medium of glutamine at the low concentrations. Cystine requirements of both cell types increase with serum concentration, probably due to the binding of half-cystine residues by the serum. Calf serum is a much more potent stimulator of cell multiplication than calf lymph, especially for the nontransformed cells. The rate of cell multiplication can be reduced by lowering the concentration of essential amino acids to the physiologic level found in body fluids, but the growth limitations can be fully compensated by simply raising the serum concentration. Growth factors may act by enhancing the utilization of amino acids, particularly of glutamine which is a required substrate for the first and chief regulatory steps of purine and pyrimidine synthesis. Lymph, which is coextensive with interstitial fluid in vivo, is poor in growth factors for the nontransformed BLAB/3T3 cells as well as for recently explanted mouse embryo cells, which raises questions of how normal cell growth is maintained in the body. This work was supported by USPHS grant CA-15744 from the Division of Extramural Activities, National Cancer Institute; by American Cancer Society Research Development Program grant RD-231; and by The Council for Tobacco Research grant 1948.     

The fine structure of the cell coat of the peritoneal macrophage and its role in the recognition of foreign material

Summary The mouse peritoneal macrophage has a prominent cell coat, clearly demonstrated by ruthenium red staining, probably containing significant amounts of acidic mucosubstances and tightly adherent to the cell membrane. Aldehyde-fixed autologous red cells are recognized at the level of a protein layer which can be readily removed without removing the cell coat.We are grateful to Professor R. Barer for his advice and criticism. The work was supported by a grant from the Medical Research Council. The three junior authors took part in this project as part of the B. Sc. Honours course in the Department. Much of the technical work was carried out by Miss Anne Edwards.     

Induction of bone,cartilage and hemopoietic tissue by subcutaneously implanted tissue diaphragms

Summary The subcutaneous implantation of appropriate tissue diaphragms (short, broad glass cylinders) rather regularly induces the formation of bones provided with marrow cavities and junction cartilage plates, in the rat.Bone formation, unlike the development of hemopoietic tissue, can be prevented, under these circumstances, by topical blockade of the phagocyte system with carbon particles.The role of nonspecific topical stress factors, in the induction of such highly specific structural transformations, has been discussed.These investigations were supported by Grant No. A-1641 (R1) from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service, by the U.S. Army Medical Research and Development Command, Department of the Army, Contract No. DA-49-007-MD-2039, and by a grant from theGustavus andLouise Pfeifer Research Foundation.     

The occurrence of intramitochondrial granules in nerve cells

Summary The mitochondria of spinal cord motoneurons of normal rats have been studied and compared with those of animals given 2.5 mg/kg of strychnine sulfate. The mitochondria in the motoneurons of the treated animals exhibited opaque granules in the matrix. Such granules were observed only when calcium chloride was present in the fixing media and were not present in any other component of the nervous tissue of the spinal cord. Granules were not seen in the mitochondria of motoneurons of untreated control animals. The possible function of these granules in nerve cell metabolism is discussed.Research supported by USPHS Grant NB 05665, 00690 and AM 08222.Recipient of Career Research Development Award 1-K3-GM 28064.     

Lack of coordination between glucose-6-phosphatase and gamma glutamyltranspeptidase activities in rat hepatocytes maintained in primary culture

Summary Glucose-6-phosphatase activity decreases whereas gamma glutamyltranspeptidase activity increases during hepatocarcinogenesis and the maintenance of hepatocytes in primary culture. This report describes the effect of culture conditions that are known to preserve hepatic glucose-6-phosphatase activity on gamma glutamyltranspeptidase activity. The results indicate that the regulation of glucose-6-phosphatase and gamma glutamyltranspeptidase activities is not coordinated in primary cultures of hepatocytes. This work was supported by a grant from the USPHS, NIH grant AG00439 awarded to Dr. Christopher C. Widnell and a Category I Research Development Award from the University of Pittsburgh to Dr. Kathleen Dobrosielski-Vergona. Editor's Statement Information communicated in this article contributes to a greater understanding of the mechanisms regulating liver cell metabolism and provides some further insight concerning the complexity of the controls involved in vitro, and presumably in vivo. David W. Barnes     

A quantitative electron histochemical estimation of ruthenium red binding to heparin in mast cell granules

Summary Mast cell granules were shown to contain an apparently branched meshwork of fibers, which had a diameter of about 240 Å and a denser core of about 20–30 Å. Mast cell granules from rats were found to have a median weight of 39×10^–14 g after critical-point drying. Their dry mass increased 23% when stained with ruthenium red at pH 5.0. The staining reaction was found to be stoichiometric, and the bulk of the stain appeared to be taken up by heparin in a molar relationship of one ruthenium red cationic complex per sulfate group of heparin. The uptake of the stain was largely localized to the cores of the granule fibers, which after staining appeared double contoured. These findings suggest that mast cell heparin is integrated into the fibrous structure of the mast cell granules.This project was supported in part by Grant No. P-259-H from the American Cancer Society.The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Enhancement of human amniotic cell growth by ficoll-paque gradient fractionation

Summary Ficoll-Paque isopycnic centrifugation was used as a preparative procdure for amniotic fluid (AF) cells prior to tissue culture. This technique serves to reduce contaminating erythrocytes and also enhances cell growth or mitotic indices. The technique described in this report yields three subfractions designatedaas a turbid interphase layer (F-2), a middle cell layer (F-3), and a bottom pellet (F-4). The middle cell layer (F-3) demonstrated better cell growth and higher mitotic index than any of the other fractions or control unfractionated amniotic fluid cells. The use of Ficoll-Paque isopycnic preparative centrifugation of amniotic fluid cells is a valuable adjunct in cell culture for cytogenetic analysis. This may be especially true when amniotic fluid contains large numbers of erythrocytes. Hsiao-chen Chang was supported by National Research Service Award 1 F32 AM HD 05887-01 from the National Institute of Arthritis, Metabolism, and Digestive Diseases; U. S. Public Health Service, National Institutes of Health. This work was supported in part by USPHS Human Biochemical Genetics Program (G177 17702-9) and the State of California, Department of Public Health, Prenatal Diagnosis Program (79-00016).     

Preparation of delipidized serum protein for use in cell culture systems

Summary A rapid procedure for the preparation of delipidized serum protein is described. The delipidized protein can be used for the maintenance and growth of tissue culture cells in a lipid-free environment. The extraction procedure greatly reduces all serum lipid classes and the delipidized protein supports the growth of a variety of cells in culture. This research was supported in part by USPHS research grants HL-09103 and HL-16058 from the National Heart and Lung Institute and RR-05540 from the Division of Research Resources. This work was done during tenure as Established Investigator of the American Heart Association (G.H.R.).     

Human red blood cell polymorphisms and malaria

Genetic factors are a major determinant of child survival in malaria endemic countries. Identifying which genes are involved and how they affect the malaria disease risk potentially offers a powerful mechanism through which to learn more about the host-parasite relationship. The past few years have seen significant progress towards achieving this goal for some of the best-known malaria resistance genes that determine the structure or function of red blood cells: Gerbich blood group antigen negativity; polymorphisms of the complement receptor genes (most notably CR1); Southeast Asian ovalocytosis; pyruvate kinase deficiency; haemoglobin E; the sickle cell trait; and alpha-thalassaemia are all examples. The challenge for the future must be to translate such advances into fresh approaches to the prevention and treatment of malaria.     

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Carotid body catecholamines

Summary Histochemical studies were made in the cat's carotid body using two fluorescence techniques: the formaldehyde condensation (HCOH) method and the trihydroxyindole (THI) method. A number of pharmacologic agents known to alter catecholamine metabolism or binding were used to evaluate their effect on the amines found in the glomus cells. After treatment with reserpine, both histochemical techniques showed a reduction and eventual disappearance of fluorescence from the glomus cells. Treatment with a monoamine oxidase inhibitor (iproniazid) or with dopamine beta hydroxylase inhibitor (disulfiran) enhanced the glomus cell fluorescence. The observed increase was greater with the THI than with the HCOH technique. A few yellow fluorescent cells were found following a combination of reserpine and iproniazid treatments. A reduction in fluorescence with both techniques was obtained following DOPA decarboxylase inhibitors (MK-485). It is concluded that some of the glomus cells contain only dopamine while others contain norepinephrine or a combination of norepinephrine and dopamine. In addition the presence of DOPA in some cells following treatment with pharmacologic agents may account for some of the results. Finally, the few yellow fluorescent cells found probably contain 5-hydroxytryptamine.Supported by a grant from the Life Insurance Medical Research Fund, USPHS General Research Support Grant and USPHS Career Development Award (K3-GM-15, 457).     

QTLs for murine red blood cell parameters in LG/J and SM/J F2 and advanced intercross lines

Red blood cells are essential for oxygen transport and other physiologic processes. Red cell characteristics are typically determined by complete blood counts which measure parameters such as hemoglobin levels and mean corpuscular volumes; these parameters reflect the quality and quantity of red cells in the circulation at any particular moment. To identify the genetic determinants of red cell parameters, we performed genome-wide association analysis on LG/J×SM/J F2 and F34 advanced intercross lines using single nucleotide polymorphism genotyping and a novel algorithm for mapping in the combined populations. We identified significant quantitative trait loci for red cell parameters on chromosomes 6, 7, 8, 10, 12, and 17; our use of advanced intercross lines reduced the quantitative trait loci interval width from 1.6- to 9.4-fold. Using the genomic sequences of LG/J and SM/J mice, we identified nonsynonymous coding single nucleotide polymorphisms in candidate genes residing within quantitative trait loci and performed sequence alignments and molecular modeling to gauge the potential impact of amino acid substitutions. These results should aid in the identification of genes critical for red cell physiology and metabolism and demonstrate the utility of advanced intercross lines in uncovering genetic determinants of inherited traits.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.