A novel technique for containerless protein crystallization

Heterogeneous nucleation, which is often detrimental to the production of suitable crystals for X-ray diffraction, can be induced by the contact of a crystallization sample with the walls of its supporting vessel. A novel method for creating a 'containerless' environment for the growth of protein crystals is described. Contact between the container walls and a crystallization drop is eliminated by suspending the drop between two oils of different density: one of higher and the other of lower density than that of water and the common precipitating agents. A number of proteins were crystallized in 2-10 microliters drops using this procedure. It was found that the number of crystals obtained in such suspended drops was reduced significantly compared with the number of crystals obtained in trials where the crystallization drop was situated at the bottom of a vial under a single layer of oil. This method has potential in controlling heterogeneous nucleation.     

Pharmacokinetic monitoring and dose modification of etanidazole in the RTOG 85-27 phase III head and neck trial

PURPOSE: To prospectively evaluate the pharmacokinetic monitoring and drug dose adjustment of Etanidazole (Eta) in patients treated on the RTOG randomized trial for Stage III and IV head and neck cancer. METHODS AND MATERIALS: From June, 1986 to October, 1991, 521 patients were randomized to conventional RT alone or RT plus Eta. The primary goal was to determine whether the addition of Eta to conventional radiation therapy improves local-regional control and tumor-free survival. Of the 264 patients who received Eta, 233 had their drug exposure calculated and the Eta dose and schedule adjusted accordingly to prevent the occurrence of serious peripheral neuropathy. Drug exposure was assessed using the area under the curve (AUC) for a single treatment that was calculated by the integral over time of the serum concentration of Eta. The total drug exposure (total-AUC) was estimated by multiplying the AUC by the number of drug administrations. RESULTS: Eighteen percent of patients developed Grade I and 6% developed Grade II peripheral neuropathy. There was no Grade 3 or 4 peripheral neuropathy. There is a trend for an increased risk of neuropathy by single dose AUC. The minimal difference in incidence of neuropathy by single-dose AUC was due to the use of dose and schedule modification for patients with the higher values. CONCLUSIONS: The pharmacokinetics investigated in this study confirm previous work that monitoring Eta levels, with dose adjustment, allows it to be used safely in the clinic. In a subset analysis there was a statistically significant improvement in local-regional control and survival rates for patients with N0 and N1 disease, that will require confirmation (14). However, the clinical efficacy of Eta in this trial proved to be of little overall benefit.     

Poisoning with N-3-pyridylmethyl-N'-p-nitrophenylurea (Vacor). Immunoperoxidase demonstration of beta-cell destruction

A 49-year-old woman ate N-3-pyridylmethyl-N'-p'nitrophenylurea (PNU; Vacor) and was admitted to the hospital 12 hours later with a blood glucose level of 940 mg/dl and an anion-gap metabolic acidosis. Her diabetes was successfully treated, but she contained to manifest severe orthostatic hypotension. A painless ileus developed, followed by cecal perforation and death. Immunoperoxidase staining of paraffin-embedded pancreatic tissue obtained during postmortem examination clearly demonstrated the pancreatic beta-cell destruction. Results of this same staining technique also suggested that fewer alpha cells were present.     

Characteristics of different sublines of VNK-21 cells

Comparative cytological, histological, histochemical and partially karyological studies were carried out on 8 batches of BHK-21 cell kept at different conditons. It was found that cells adapted to suspension culture differed from monolayer culture cells both morphologically and cytochemically. Suspension cells transmitted into monolayer culture displayed smaller size, changeable form, increased glycogen accumulation, decreased enzymatic activity (acid phosphatase, succinate dehydrogenase), their karyotypes tending to hypodiploidy.     

Irreversible inactivation of protein kinase C by a peptide-substrate analog

Protein kinase C (PKC) is a phospholipid-dependent isozyme family that plays a pivotal role in mammalian signal-transduction pathways that mediate cell growth and differentiation and pathological developments, such as the acquisition of drug resistance by cancer cells. Several peptide-substrate analogs have been shown to reversibly inhibit PKC with high potency and selectivity, but peptide-substrate analogs that antagonize PKC by forming a covalent complex with the enzyme have not been reported. The development of active site-directed irreversible inactivators of PKC could provide new insights into the catalytic mechanism and might ultimately lead to the design of novel therapeutics targeted at PKC. In this report, we show that the peptide-substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates PKC in a dithiothreitol-sensitive manner. The inactivation mechanism most consistent with our results is the formation of a covalent linkage between the inhibitor-peptide and the enzyme at its active-site. Limited proteolysis of PKC produces a catalytic-domain fragment that is independent of the phospholipid cofactor. RKRCLRRL antagonized the histone kinase activity of PKC and its catalytic-domain fragment with similar efficacies, achieving > 50% inactivation at an RKRCLRRL concentration of 10 microM. In contrast, RKRCLRRL analogs with single amino acid substitutions at Cys were non-inhibitory. The inactivated complex of the catalytic-domain fragment and RKRCLRRL was stable upon dilution, and the inactivation of PKC and the catalytic-domain fragment by RKRCLRRL was quenched by dithiothreitol, providing evidence that the enzyme and the synthetic peptide may be covalently linked in an inactivated complex by a disulfide bond. Substrates and substrate analogs protected the catalytic-domain fragment against inactivation by RKRCLRRL, providing evidence that inactivation entailed binding of RKRCLRRL at the active-site of the enzyme. S-Thiolation is the formation of mixed disulfides between proteins and low molecular weight thiols. PKC is thought to have a highly reactive Cys residue in its active-site, and Cys residues that are flanked by basic residues, as is the case in RKRCLRRL, display enhanced reactivity. Our results support an inactivation mechanism that entails S-thiolation of the active-site of PKC by RKRCLRRL. This is the first report of irreversible inactivation of PKC by an active site-directed peptide.