The interactions of cis- and trans-diammineplatinum compounds with 5′-guanosine monophosphate and 5′-deoxyguanosine monophosphate. A proton nmr investigation

The interactions of cis- and trans-diammineplatinum compounds with 5′-GMP and 5′-dGMP in dilute aqueous solution at neutral pH were investigated by ¹H nmr. In addition to the 1:2 Pt nucleotide complexes cis- and trans-Pt(NH₃)₂(GMP)₂, it was possible to study the formation of the 1:1 Pt-nucleotide complexes with either one coordinated water or chloride ion. At 5°C GMP reacts with a stoichiometric amount of cis-diaquodiammine-platinum to yield cis-Pt(NH₃)₂(GMP) (H₂O) as a sole reaction product. From the present results it is concluded that such a complex may play an important role as the initial reaction product between antitumor compounds like cis-Pt(NH₃)₂Cl₂ and guanine in DNA in living organisms. The coupling constant ³J(H(1′)-H(2′)) of the H(1′) sugar proton in cis-Pt(NH₃)₂(GMP)₂ is temperature dependent, indicating a conformational change in the sugar moiety.     

Kinetic studies on the cupric ion oxidation of sheep hemoglobin

The oxidation of sheep hemoglobin, in both the oxygenated and deoxygenated forms, by cuprous ions have been studied by spectrophotometric and stopped-flow techniques. Mixing of both the oxy and deoxy forms with excess Cu²⁺ leads to the rapid oxidation of the iron atoms of all four of the hem groups of the tetrameric protein, followed by the slow formation of hemichromes (low spin Fe^III forms of hemoglobin). Stopped-flow studies show that the oxidations follow simple monophasic kinetics with second-order rate constants of 65 and 310 M^?1 sec^?1 for the oxy and deoxy forms, respectively. Variable temperature studies yield Arrhenius activation energies of 43 for the oxy form and 113 kJ mole^?1 for the deoxy form. For each form of the protein the activation energy is very similar to the activation enthalpy. While the deoxy form is characterized by an activation energy and enthalpy that is more than twice the corresponding value in the oxy form. The activation entropies show highly significant differences being ?128 e.u. and 136 e.u. at 25°C for the oxy and deoxy forms, respectively.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

13C nmr studies of aurothioglucose: Ligand exchange and redox disproportionation reactions

¹³C nmr studies of gold thioglucose, AuSTg, and solutions containing added β-1-D-thioglucose, TgSH, have been conducted at PD 7.4 and interpreted in terms of complexation and ligand exchange reactions that are consistent with the known preference of gold(I) for linear two-coordinate structures. The upper limit of the half-life for ligand exchange between 0.25 M Au(STg)₂^? and TgSH at pD 7.4 is 2.2 msec. The ¹³C nmr spectra of various thioglucose derivatives have been assigned. A novel oxidation reduction reaction was discovered that leads to the formation of metallic gold and a product tentatively identified as the sulfinic acid derivative of thioglucose. The presence of sulfinic acid in AuSTg was indicated by the infrared absorption at 1050 cm^?1. The same product was formed by slow hydrolysis of thioglucose disulfide. A mechanism for the formation of the sulfinic acid derivative from AuSTg is proposed.     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

Neurological and electroencephalographic changes in newborns treated with prostaglandins E2 and E2

Six newborns with obstructive right heart lesions were examined neurologically and electroencephalographically during treatment with prostaglandin (PG) E₁ or E₂ given to maintain patency of the ductus arteriosus and to increase pulmonary blood flow. PG was administered intravenously or intraarterially in the aortic isthmus proximal to the ductus arteriosus. Besides a rise in arterial oxygen saturation, all patients had some sign of central nervous system involvement. The electroencephalogram showed minor changes suggestive of sedation. In addition, three patients in whom PG given intravenously presented various combinations of neurological abnormalities (“myoclonic jerks”, apnoeic spells, hiccup) of subcortical origin. Side-effects subsided after stopping the treatment anf posed no problem in the management of the patients. These findings confirm the usefulness and safety of the PG therapy and indicate that the intraaortic route of administration is preferable.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.