Cloning,characterization, and expression of two α-amylase genes from Aspergillus niger var. awamori

Summary Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an -amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two -amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.Smith Kline Beecham Pharmaceuticals, King of Prussia, PA, USA.     

Dihydropyridines,phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca²⁺ channel currents recorded from a holding potential of –100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca²⁺ channel current (EC₅₀: 1 M, 4 M and 40 M, respectively). At these concentrations, the drugs could also inhibit HVA Ca²⁺ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2–10 times these concentrations. The -conotoxin-GVIA-sensitive (-CTx-GVIA, N-type), -agatoxin-IVA-sensitive (-Aga-IVA, P- and Q-types) and non-L-type -CTx-GVIA-, -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that CTx-GVIA and -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca²⁺ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca²⁺ channels.     

Untersuchungen zur Ultrastruktur lympho-epithelialer Thymustumoren unter besonderer Berücksichtigung der sog. „Emperipolesis“

Summary The ultrastructure of eleven thymomas with lymphocytic predominance, one epitheloid cell thymoma and two normal human thymuses is described with special reference to Emperipolesis. All patients have had myasthenia gravis.The normal human thymus consists of three parts: outer cortex, inner cortex, and medulla. The outer cortex contains mainly lymphoblasts and Metcalf's macrophages within the so-called Clark-packet's. The inner cortex consists mainly lymphocytes and interdigitating reticulum cells, and the medulla of epithelial cells, lymphocytes and Hassall's corpuscles.In all cases of lympho-epithelial thymoma and in normal human thymuses there are enormous interdigitations between epithelial (tumor) cells, lymphocytes and macrophages. The epitheloid cell thymomas also show findings which suggest an epithelial cell interaction. We have not found intact lymphocytes inside the cytoplasm of normal and/or tumor epithelial cells, macrophages or interdigitating reticulum cells.The intracellular existence of intact lymphocytes has been termed Emperipolesis by Humble, Jayne, and Pulvertaft, meaning internal wandering. These investigations indicate that Emperipolesis is not an adequate term for cellular interaction in normal human thymuses and thymomas. A false impression of intraepithelial location of thymic lymphocytes is created by two-dimensional sections of complex thymic structure. These ultrastructural studies revealed damage to lymphocytes only in macrophages with lymphocytolysis within these cells and accumulation of numerous heterophagic vacuoles containing fragments of lymphocytic debris within them.

FrÄulein C. Schürmann danke ich für die gute technische Assistenz, Herrn Priv.-Doz. Dr. med. R. W. Ch. Janzen, Neurologische Klinik der UniversitÄt Hamburg, für die klinischen Daten der Myasthenie-Patienten     

Neutralization of Transforming Growth Factor β1 Augments Hepatitis C Virus-Specific Cytotoxic T Lymphocyte Induction in Vitro

In hepatitis C virus (HCV) infection, TGF-1 is upregulated in the liver and may be involved in the pathogenesis of chronic liver disease. TGF-1 is also produced by activated T cells and acts as a potent immunosuppressor. The aim of this study was to investigate the roles of TGF-1 in HCV-specific cytotoxic T lymphocyte (CTL) induction and enhance their killer activity by TGF-1 modulation. We generated anti-HCV CTL from peripheral blood mononuclear cells from HLA-A2 patients under stimulation with the HCV-core peptide having the HLA-A2.1 binding motif. The lytic activities of CTL or precursor frequency (CTLpf) generated with or without anti-TGF-p antibody were compared. To optimize the IL-2 dose for CTL induction, low (50 U/ml) and high (500 U/ml) doses were tested and the lytic activities were compared. TGF-1 amounts in the supernatants were assessed by enzyme-linked immunosorbent assay and by their growth inhibitory effect on mink lung epithelial cells. CTL activity was enhanced by anti-TGF- antibody in a dose-dependent manner but CTLpf did not significantly change. A high dose of IL-2 reduced the activity to 45% of that observed with a low dose, whereas TGF-1 increased as the dose of IL-2 increased. Exogenous IL-10 reversed the inhibitory effect of a high dose of IL-2 on the killing activity by reducing TGF-1 mRNA expression in T cells and its production. These results demonstrated that endogenous TGF-1 is an autocrine suppressor in CTL induction in vitro. Therefore, the blockade of endogenous TGF-1 could enhance the killing potential of anti-HCV CTL.     

Proteinase — Antiproteinase imbalance in meningitis: Determination of α 1 proteinase inhibitor (α 1PI), elastase — α-1PI complex,and elastase inhibition capacity in cerebrospinal fluid

Summary Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness.Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions.To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase 1-proteinase inhibitor (elastase- 1PI), 1-proteinase inhibitor ( 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-214 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month–15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months–15 years. The concentration of elastase- 1PI in the BM group (median 552 g/l) was significantly higher than in either the NBM group (median 30 g/l,p<0.01) or the control group (median 30 g/l,p<0.01). Similarly, the 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l,p<0.025) or the control group (median 6.3 mg/l,p<0.001). The concentration of elastase- 1PI shows a significant correlation with the duration of the infectious symptoms before admission to the hospital (r=0.51,p<0.02), but not with the number of neutrophil granulocytesr=0.23, p=0.21).Free elastolytic capacity in CSF could be demonstrated in 4 patients: 1 with BM, 2 with NBM, and 1 with pertussis pneumonia and enzephalitis.The measured insufficiency of the proteinase-antiproteinase system may indicate high-risk patients in need of additional anti-inflammatory therapy, e.g., with corticosteroids, during the initial phase of meningitis.Abbreviations 1PI 1-proteinase inhibitor, 1-antitrypsin - elastase- 1PI complex elastase- 1-proteinase inhibitor complex - EIC elastase inhibition capacity - BM group: bacterial meningitis - NBM group: non-bacterial meningitis - CSF cerebrospinal fluid     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Role of adrenergic structures in functional control over the cerebral circulation

An investigation of the cerebral circulation by the thermoelectric method showed that stimulation of the cervical sympathetic nerve leads to considerable changes in the blood supply to the brain. The changes in blood flow are biphasic in character: An initial small increase is followed by a decrease below the original level. Pharmacological analysis with and adrenoblockers showed that the constrictor response of the cerebral vessels is due to excitation of-adrenergic structures and the dilator response to excitation of-adrenergic structures. A possible mechanism of these changes is postulated.Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 9–12, January, 1976.     

The isolated frog skin epithelium: Presence of α and β adrenergic receptors regulating active sodium transport and water permeability

Summary The effects of stimulation of and adrenergic receptors on short circuit current (S.C.C.), Na⁺ and Cl^– fluxes and osmotic water permeability were studied on isolated frog skin epithelial layers separated from the dermis.Low norepinephrine doses (final concentrations in the incubation medium ranging from 5×10^–9 to 10^–8 M) produced increased water permeability and S.C.C. The latter was entirely accounted for by an increase in the active Na⁺ influx. Na⁺ outflux and Cl^– fluxes were not modified. Both these effects disappeared after treatment with the blocking agent, Propranolol. Higher norepinephrine doses (final concentrations: 10^–7 to 10^–6 M) produced: 1. an increase in water permeability lower than that produced by low doses, the highest doses failing to increase water permeability, and 2. a triphasic change in S.C.C.: after an initial increase, S.C.C. dropped to its resting value and then rose again to a sustained value. Na⁺ and Cl^– flux measurements showed that the variation in S.C.C. reflected variations in active Na⁺ transport. When the same high norepinephrine doses were applied after treatment with the blocking agent Phentolamine, the effects observed were identical to those obtained with low doses.On blocked preparations, large doses of norepinephrine inhibited the water permeability and sodium transport increases induced by theophylline or oxytocin but did not modify those induced by 35-cyclic AMP. The inhibition was suppressed after blocking receptors.From the foregoing, it was concluded that both and adrenergic receptors are present in frog skin epithelial cells and are involved in the regulation of water and sodium permeability.It is suggested that the inhibitory effect of stimulation resulted from the inhibition of cyclic-AMP generating system, the activity of which is under the positive control effect of oxytocin and stimulation.     

Regulated overproduction of α-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis

Summary High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called SwARS1. The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5–10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned -amylase gene from S. occidentalis secreted about five times more -amylase than cells without additional copies of the -amylase gene. Both the chromosomal copy and the plasmid-carried copies of the -amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.     

γδ T Lymphocytosis Associated with Common Variable Immunodeficiency1

We present the case of a 28-year-old Caucasian female with common variable immunodeficiency (CVID) since age 5 who had a long history of hospitalizations for unexplained fevers and pulmonary infiltrates. The patient developed mild lymphocytosis 7 months prior to our evaluation. Flow cytometry of peripheral blood revealed an expansion of T lymphocytes, mild CD4 T lymphocytopenia, and a reduced CD4/CD8 ratio (0.2). Two subpopulations of T lymphocytes were found (CD3⁺/CD4^–/CD8⁺, 47%; CD3⁺/CD4^–/CD8^–, 53%), the vast majority of which expressed V-1. An infectious cause for the patient's T lymphocytosis could not be found. The sputum was chronically colonized with Staphylococcus aureus, and the organism produced TSST-1 in vitro. A bronchoalveolar lavage (BAL) revealed marked lymphocytosis, but T lymphocytes were not overrepresented in the BAL. Lymphocyte functional studies revealed poor proliferative responses to mitogens and staphylococcal superantigens and diminished cytokine production. V-1 T lymphocytes from the patient's blood were not expanded in vitro in response to staphylococcal superantigens. TCR gene rearrangement studies confirmed the presence of J and J1 clonal rearrangements accounting for only a small subpopulation of the T lymphocytes. These studies were repeated 5 months later and were unchanged. A bone marrow biopsy was negative for leukemia. Hence, the cause of the patient's T lymphocytosis could not be determined despite evaluation for underlying malignancy, occult infection, or superantigen-driven stimulation. The patient ultimately died of progressive respiratory insufficiency. The state of current knowledge regarding T lymphocytosis, decreased production of T lymphocytes, and a low CD4/CD8 ratio in association with CVID is discussed.     

Differential secretion of TNF-α and IFN-γ by human peripheral blood-derived NK subsets and association with functional maturation

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF- and IFN- by both the binder and the killer subsets but not by the free subset. IFN- activated the secretion of IFN- only, whereas IL-2 activated the secretion of both TNF- and IFN- by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF- and IFN- secretion in both the binder and the killer subsets, though IFN- secretion was more pronounced in the binder subset. Activation of TNF- and IFN- secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF- and IFN-, whereas the free NK subset secretes little or no TNF- and IFN- following activation. These data suggest that the ability of NK cells to secrete TNF- and IFN- following activation correlates with the functional stage of maturation of NK cells.     

The importance of the perfusion pressure in the coronary arteries for the contractility and the oxygen consumption of the heart

Summary The influence of increasing perfusion pressure in the coronary arteries on cardiac contractility and oxygen consumption was studied in isovolumetrically working, empty beating, and potassium arrested guinea-pig hearts. Raising the coronary perfusion pressure from 60 to 80 cm H₂O increased left ventricular peak systolic pressure by 19.4±2.8%, coronary flow by 47.7±8.0% and oxygen consumption by 32.7±6.2% (N=17).At a constant perfusion pressure hypoxia increased the coronary flow by 111.8±16.4% (N=19), but did not alter the contractility of the heart or its oxygen consumption.Increasing pressure without changing flow, raised pressure in the left ventricle by 26.5±4.8% and myocardial oxygen consumption by 20.3±3.4% (N=14). The end-diastolic pressure and the heart rate remained unchanged in all experiments.From these findings it may be concluded that the pressure in the coronary vessels — independent of the flow rate — exercises a positive inotropic effect, thereby increasing metabolism. This can be explained by increased fibre tension, caused by an extension of the coronary vessels, the so-called garden-hose-effect.Partially delivered during the 31st Meeting of the Deutsche Physiologische Gesellschaft in Würzburg, 1966 [2].     

Identification of an Adhesion Molecule Expressed on Adult T Cell Leukemia Cells Derived from a Patient with Gastrointestinal Involvement: Implication for a Possible Role of Integrin β7 in Leukemic Cell Infiltration into Intestinal Mucosa

Patients with adult T cell leukemia (ATL) often manifest leukemic cell infiltration into various organs such as lung, liver, skin, and gut. To analyze the mechanism of intestinal infiltration of ATL cells, we made mAbs against ATL-43T, a human T cell line derived from an ATL patient with severe intestinal mucosal infiltration. One of the mAbs, named H920, was noted for a high and relatively specific reactivity with ATL-43T. Molecular cloning was done to identify this molecule and disclosed that the Ag molecule was identical to integrin 7. Since integrin 7 and its ligand MAdCAM-1 had been reported to mediate homing of lymphocytes to endothelial cells in intestinal mucosa, we next examined whether ATL-43T cells could adhere to MAdCAM-1⁺ cells. Human MAdCAM-1 transfectants of MMCE, a mouse epithelial cell line, were made and used to evaluate cell adhesion mediated by integrin 7 and MAdCAM-1. Considerable levels of cell adhesion were observed between ATL-43T and the transfectant cells, which was inhibited by H920 mAb in a dose-dependent manner. Furthermore, peripheral blood leukemic cells or lymphoma cells from 10 ATL patients were examined for expression of integrin 7 with regard to organ involvement. Samples from three patients with gastrointestinal tract involvement showed considerably higher expression of integrin 7. These results suggest that integrin 7 may play a role in adhesion and subsequent infiltration of a certain type of ATL cells into intestinal mucosa.     

Liberation ofβ-lipoprotein,alkaline phosphatase,and 5′-nucleotidase from platelets in response to aggregation induced by adrenalin

During aggregation induced by adrenalin,-lipoprotein, alkaline phosphatase, 5-nucleotidase, and factor 3 are liberated from the platelets. The electrophoretic mobility of platelet alkaline phosphatase is the same as that of the-lipoprotein. This suggests that the-lipoprotein, alkaline phosphatase, and 5-nucleotidase are structural components of platelet material carrying the factor 3 activity.Department of Biochemistry, S. V. Kurashov Medical Institute, Kazan'. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No, 5, pp. 545–547, May, 1976     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leul gene

Summary The leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu ^-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus -IPM (isopropylmalate) isomerase.     

The mitochondrial genome of fertile maize (Zea mays L.) contains two copies of the gene encoding the α-subunit of the F1-ATPase

Summary In contrast to the situation in animals and fungi the -subunit of the mitochondrial F₁-ATPase is encoded by two identical mitochondrial genes (ATP A) in male fertile maize (Zea mays L.). Cytoplasmic male sterile (T, C and S) maize mitochondrial genomes only contain a single copy of the gene. Sequence analysis reveals that the uninterrupted coding region of both copies of the gene is 1,524 by long and encodes a polypeptide of 508 amino acids with a molecular weight of 55,117. The predicted amino acid sequence shares over 60% homology with the nuclear encoded -subunit from yeast and bovine ATPases and approx. 50% with the corresponding chloroplast and bacterial polypeptides.     

Control of metastatic properties of BL6 melanoma cells by H-2Kb gene: immunological and nonimmunological mechanisms

The effect of class I H-2 antigen expression on the metastatic properties of BL6 melanoma cells was investigated. The BL6-8 clone isolated from the highly metastatic BL6 melanoma did not express H-2K b gene. Following transfection with the H-2K^b gene, BL6-8 cells displayed a low metastatic potential in the immunocompetent as well as immunosuppressed (X-irradiated) or triple-immunodeficient mice with impaired T, B and natural killer (NK) cells function. The expression of H-2K^b gene and the low metastatic ability of transfected BL6 melanoma cells were associated with appearance of cell membrane soybean agglutinin (SBA) and Griffonia simplicifolia 1B₄ (GS1B₄) lectin-binding carbohydrataes. These alterations in cell surface carbohydrates were found to be a result of reduction in sialylation of SBA binding sites and upregulation of the 1.3 galactosyltransferase (1.3GT) gene. To assess the importance of H-2K^b-induced alterations in cell surface carbohydrates for metastasis formation, BL6-8 melanoma cells were transfected with H-2K^b gene without neo^r gene cotransfection and selected for adherence to SBA-lectin-conjugated agarose beads. The transfected clones that expressed SBA and GS1B₄ lectin-binding carbohydrates were low metastatic. Further analysis of these clones showed that presence of SBA and GS1B₄ lectin-binding carbohydrates rather than expression of H-2K^b molecules per se might be responsible for low metastatic potentials of H-2K^b-transfected cells in the immunocompromized mice. Studies of the possible mechanisms responsible for low metastatic ability of H-2K^b-transfected melanoma cells revealed that these cells displayed a reduced ability to adhere to murine pulmonary endothelial cells as well as to laminin and collagen IV. We hypothesized that the observed nonimmunological effects of H-2K^b gene in BL6 melanoma cells is a result of an interaction between the H-2K^b gene and B16 melanoma-specific ecotropic retrovirus. It results in inhibition of this retrovirus production with consecutive alteration in the expression of cellular genes controlling cell surface glycosylation and adhesion properties essential for the metastatic phenotype of BL6 melanoma.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.