Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning

Seminal vesicle secretory protein IV of a mouse has been isolated, and the cDNA coding for its mRNA has been cloned and sequenced. The 556-nucleotides encode 16 amino acid signal peptides and 92 residues of mature protein. Considerable homology between mouse and rat SVS IV cDNA was found. In the leader peptide and 3'-noncoding region there is 92% and 85% homology, respectively. The other regional homologies are 86% for the first 12, 68.5% for the last 35, and 40% for the middle 44 amino acids. The expression of mouse SVS IV mRNA is under the control of androgen. Administration of testosterone to castrated mice resulted in induction of the mRNA level to 50% of the mature male in 96 h of hormone treatment. Secretion of the protein after testosterone injection follows a similar pattern.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.     

Thermotolerance of isolated mitochondria associated with heat shock proteins

Mitochondria isolated from 2-day-old etiolated soybean (Glycine max) seedlings which had been subjected to various heat shock treatments, i.e. (A) 28°C (2 h), (B) 38°C (2 h), (C) 38°C (2 h)-42.5°C (0.5 h), and (D) 38°C (2 h)-42.5°C (0.5 h)-28°C (4 h), were monitored for O₂ uptake using an oxygen electrode. Mitochondria isolated after all four heat shock treatments were active in O₂ consumption at 28°C in response to succinate and ADP (derived P/O ratios were 1.6, 1.7, 1.3, and 1.3, respectively.) The mitochondria from all four treatments were also active in O₂ uptake at 42.5°C. However, only mitochondria isolated after treatment (C) were tightly coupling at 42.5°C (derived ADP/O ratio was about 1.4). Combined with our earlier findings on the subcellular localization of heat shock proteins, our present data demonstrate that association of heat shock proteins with mitochondria by treatment (C) enables them to phosphorylate at 42.5°C (i.e. they become thermotolerant). Isolated mitochondria from treatment (C) and treatment (A) were compared by electron microscopy. They appeared to be very similar and no significant ultrastructural differences were noted.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

Combined second and third toe transfer

Historically, restoration of hand function following multiple digital amputation has been unsatisfactory. The evolution of digital reconstruction with toe transfer has enabled surgeons to reestablish prehension in these severely injured hands. A 4-year experience with 26 consecutive combined second and third toe transfers to replace missing adjacent fingers was reviewed in order to delineate the indications and technical considerations and to emphasize prevention of donor-site complications. Combined second and third toe transfer is reserved for adjacent finger amputations proximal to the digital web space with remaining fingers no longer than the small finger. Radial amputations are replaced with contralateral combined toe units, while ipsilateral toes are more ideal for ulnar amputations. Limited dorsal and plantar skin flaps extending only to the midpoint of the first and third digital web spaces allow for direct donor-site closure and uncomplicated healing. Maintenance of the plantar metatarsal arch by avoiding metatarsal shaft osteotomies or bone grafting-shortened metatarsals eliminates potential gait disturbances. When properly applied in selected patients, this single-stage microsurgical procedure can restore prehensile function, improve the appearance of the hand with multiple digital amputations, and preserve near-normal donor-foot function.     

More monensin-sensitive, ammonia-producing bacteria from the rumen

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.     

Factor IXPortland: a nonsense mutation (CGA to TGA) resulting in hemophilia B

Male siblings with severe hemophilia b were studied for the molecular defect responsible for their disorder. To define the precise DNA alteration, a 362-bp fragment in the first part of exon VIII of the factor IX gene was amplified and sequenced. A single-base-pair substitution of C----T at the nucleotide sequence 30875 was found which resulted in a nonsense mutation (TGA) and terminated the protein synthesis of factor IX at amino acid residue 252. The single-base change occurred as a classic CG dinucleotide alteration to TG (or CA), a common mechanism for point mutations in mammals.