Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

Growth of mouse embryos in bicarbonate media buffered by carbon dioxide,hepes, or phosphate

The purpose of this study was to determine if mouse embryos could be grown successfully in a culture medium devoid of the carbon dioxide phase (CO₂). Mouse embryos fertilized in vivo were collected and cultured in Hepes medium with and without bicarbonate (HCO₃ ^–) and a phosphate medium with and without HCO₃ ^–. In these experiments no CO₂ gas phase was used. Further embryos were cultured in Whittingham's modified Tyrode's (T6) medium with a CO₂ gas phase and served as controls. The degree of embryonic development was noted. Surviving blastocysts were transferred to the uteri of pseudopregnant mice and delivery at term was allowed to occur. There was no significant difference in the degree of embryonic development in those embryos cultured in T6 or Hepes medium (+HCO₃ ^–) or in the number of live offspring obtained when these blastocysts were placed within the mouse uterus. Although embryonic development apparently proceeded successfully in the phosphate (+HCO₃ ^–) medium, none of these blastocysts survived when transferred to mouse uteri. No embryonic growth occurred in either the Hepes or phosphate media which were devoid of HCO₃ ^–. It appears that a Hepes medium containing HCO₃ ^–, which uses no CO₂ gas phase, is as effective as T6 medium, which uses a gas phase, in supporting in vitro mouse embryonic growth.     

Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug-DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed 'classical' pattern observed for intercalation, as proved by the DNA topoisomerase I-unwinding experiments, with relatively weak binding affinity (K(i)=1.2 x 10(5)M(-1)), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 10(5)M(-1) and binding site size of 2-3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug-DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.     

Plasma membrane potential of lymphocytes from ataxia telangiectasia patients

The plasma membrane potential of lymphocytes prepared from ataxia telangiectasia (AT) patients and normal subjects was assessed using the optical indicator bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethineoxonol (oxonol-V). AT lymphocytes had a potential of -46 +/- 9 mV and normal lymphocytes had a potential of -63 +/- 4 mV. The intracellular cation content (Na+ and K+) of AT and normal lymphocytes was similar. AT and normal lymphocytes were both depolarized by extracellular K+ and to a similar extent. This study indicates that one feature characterizing ataxia telangiectasia is a modification of the ability of the lymphocyte cell membrane to sustain a normal membrane potential.     

In vitro evaluation of phosphate,bicarbonate, and hepes buffered storage solutions on hypothermic injury to immature myocytes

Summary In this study we evaluated cardiac myocyte viability and function under hypothermic conditions using three types of buffer solutions: phosphate buffer solution (PBS), Krebs-Henseleit bicarbonate buffer solution (KHB), and Hepes buffered minimum salt solution (MSS). As a control, normal saline solution (NSS) was used. Cardiac myocytes were isolated from neonatal rat ventricles. Myocytes (12.5 × 10⁵ myocytes/culture flask) were then incubated at 4°C for 6, 12, 18, and 24 hours in various buffer solutions. After each incubation time, CPK and LDH were measured. The myocytes were then incubated for an additional 24 hours at 37°C to evaluate the recovery of the myocyte beating rate. Group MSS had a significantly better beating rate recovery than group NSS (control) after 18 hours (MSS, 32.7%, NSS, 0.0% of control; i.e., beating rate prior to hypothermic incubation). In contrast, group KHB showed a significantly lower recovery ratio than group NSS at 12 hours (41.0%, 78.8%, respectively), and the lowest recovery was observed in group PBS beginning at 6 hours of hypothermic incubation (27.6%). Group MSS significantly suppressed the release of CPK and LDH compared to group NSS at 24 hours (MSS, 246.7 and 440.2 mIU/flask; NSS, 369.7 and 821.3 mIU/flask, respectively). In contrast, groups PBS and KHB showed significantly increased CPK and LDH levels compared to group NSS after 12 hours (PBS, 388.6 and 721.4 mIU/flask; KHB, 340.5 and 540.5 mIU/flask; NSS, 91.5 and 222.7 mIU/flask, respectively). In conclusion, Hepes buffer has cytoprotective characteristics that may be suitable for long-term hypothermic preservation of immature myocardium compared to phosphate or bicarbonate buffer.     

9-Deazaadenosine—A new potent antitumor agent

9-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, ic₅₀ values ranged from 1.1 to 8.5 × 10^?8 M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10-^?5M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [³H]uridine into RNA and of [³H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.     

Uptake of chromium by rat liver mitochondria

Isolated rat liver mitochondria rapidly accumulate chromate (1.2 μM ⁵¹CrO₄^2?) to about 0.25–0.30 nmol Cr/mg protein. The relative uptake decreases with increasing chromate doses. Chromate uptake decreases when pH is raised from 7.0 to 7.5. N-ethylmaleimide (0.25 mM) and butylmalonate (5 mM) inhibit chromate uptake to 70% and 30% of control values, respectively, whereas mersalyl (40 nmol/mg protein) causes an inhibition of > 95%. Both sulphate and phosphate decrease mitochondrial chromate uptake, the former being more effective in lower doses (5 mM). These results indicate that transport of chromate is mediated both on the dicarboxylate and the phosphate carrier. The extensive mitochondrial chromium accumulation can be explained by trapping of chromium, probably by reduction of chromate to the trivalent form, within the mitochondria. Release of chromium after chromate loading was seen after 15 min. Added after chromate loading, mersalyl partly prevents this release. Trivalent chromium as ⁵¹CrCl₃ is taken up to a much lower degree than hexavalent chromium as ⁵¹CrO₄^2?. The presence of glutathione (5 mM) reduces the uptake both of ⁵¹Cr-III and ⁵¹Cr-VI, indicating extramitochondrial reduction of Cr-VI to Cr-III and subsequent binding to GSH.     

Content and release of acetylcholinesterase in skeletal muscle of rats with experimental autoimmune myasthenia gravis

The effects of experimental autoimmune myasthenia gravis (EAMG) on acetylcholinesterase (AChE) were investigated in diaphragms of adult female Lewis rats. Both total AChE activity per muscle and release of enzyme activity during a 3-h incubation in vitro were measured. Two groups of myasthenic animals were used. Acute EAMG was induced by intravenous injection 48 h earlier with a syngeneic monoclonal autoantibody against the nicotinic acetylcholine receptor (AChR) of rat skeletal muscle; age- and weight-matched controls received a monoclonal anti-AChR antibody nonreactive with mammalian muscle. Chronic EAMG was induced by immunization 4 weeks earlier with AChR purified from Torpedo electroplax; controls received only adjuvants. When preparations from rats with acute or chronic EAMG were compared with the appropriate controls, no statistically significant differences in content or release of AChE activity were detected. Neither was there any change in the relative amounts of the various molecular forms of AChE in samples from animals with chronic EAMG. We conclude that the structural and functional changes arising in EAMG are highly specific for the acetylcholine receptor and associated elements of the neuromuscular junction, but have little impact on the biology of AChE.