Sequence specificity of mutation induced by the anti-tumor drug cisplatin in the CHO aprt gene

Cis-diammine dichloroplatinum (cisplatin) is an effective anticancerdrug which forms adducts with DNA, in both bacterial and mammaliancells. It is suspected of producing tumors as well. To determinethe molecular nature of geneti alterations induced by cisplatin,we cloned and sequenced cisplatin-induced mutants in the adeninephosphoribosyl-transferase (aprt) gene of Cinese hamster ovary(CHO) cells. Mutation by cisplatin appears to be targeted asthe sites of mutation are consistent with the known bindingspecificity of cisplatin. Many mutations occur at or proximalto the sequence 5'-AGG-3' and 5'-GAG-3' and include transversions,transitions, frameshifts and short deletions and duplications.Several double changes were also observed. No major rearrangementswere recovered in our collection. At several locations, a numberof mutants were found to be clustered within a small targetregion, but unlike traditional hotspots, tese represent diversechanges occurring in a localized region of a few base pairs.     

Stimulation of in vitro myelin synthesis by microglia

Central nervous system myelin is elaborated by oligodendrocytes, which have been studied extensively in cell culture. Dissociated brain cultures allow in vitro analysis of events in myelinogenesis, including cell-cell interactions. Microglia, the primary phagocytic cell of the central nervous system, appear in developing fiber tracts prior to the onset of myelination in vivo. To gain insight into potential oligodendrocytemicroglial interactions during development, these cells were co-cultured and various parameters of myelin synthesis were measured. In co-culture, microglia stimulated the synthesis of sulfatide, a myelin-specific galactolipid, in oligodendrocytes, as well as the expression of the myelin-specific proteins myelin basic protein and proteolipid protein. Activity of the oligodendrocyte cytoplasm-specific enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase was not elevated, suggesting that the effects of microglia were not due to stimulation of oligodendrocyte proliferation. This was confirmed by the inability of microglia to induce significant DNA synthesis. Conditioned medium from cultured microglia provided a similar stimulatory activity, suggesting that the increase in myelin synthesis does not require contact between oligodendrocytes and microglia. These findings suggest a stimulatory role for microglia during myelinogenesis. © 1994 Wiley-Liss, Inc.     

X-linked muscular atrophy and the androgen receptor

X-linked muscular atrophy is a form of adult-onset, usually slowly progressive spinal and bulbar motor neuron degenerative disease that is uniquely associated with male hypogonadism. The mutation responsible for this syndrome is expansion of the trinucleotide repeat—cytosine (C), adenine (A), guanine (G)—in a 5′-translated portion of the androgen receptor (AR) gene from a normal, polymorphic length of n = 11–31 to n ≥ 40. The resulting androgen receptor (AR) protein has an expanded polyglutamine tract in its NH₂-terminal modulatory domain, and is postulated to lose a basic, intrinsic function that causes a mild form of androgen insensitivity; however, almost certainly, it also gains a novel, extrinsic function that is selectively neuronotoxic. The unexplained mechanism that culminates in this form of neuronspecific death is the prototype for three different adult-onset neuronopathies that are caused by (CAG)_n expansions in other genes.     

Focal lung uptake of Gallium-67 in patients with acquired immunodeficiency syndrome secondary to pneumocystis carinii pneumonia

It is generally accepted that the lung uptake of ⁶⁷Ga in patients with pneumocystis carinii pneumonia (PCP) is diffuse and bilateral. Three cases of focal lung uptake of ⁶⁷Ga in AIDS patients with PCP but without other opportunistic infection are described. While focal lung uptake is characteristic of opportunistic infections other than PCP, we wish to emphasize that focal uptake of gallium in the chest does not rule out PCP and may represent its earliest stage of presentation.     

A new approach to massive blood transfusion during pediatric liver resection

Morbidity and death during liver resection in children are due to hemorrhage and the consequences of massive transfusion. To overcome these problems, a new rapid method of blood transfusion was used in four children (8 to 35 months, 8.6 to 13 kg) undergoing extensive hepatic resection for tumor (tumor weight, 440 to 1625 gm). The rapid infusion device consisted of a roller pump and a bubble oxygenator-warmer circuit primed with washed packed red cells resuspended in fresh-frozen plasma and calcium-free balanced salt solution (Plasmalyte). The infusate was warmed, oxygenated, and buffered before it was administered. An average of 5130 ml per patient of this reconstituted blood was infused at an average rate of 122 +/- 45 ml/min, with peak infusion rates sometimes as great as 1 L/min. Cardiac output, pulmonary artery wedge pressure, body temperature, urine output, blood gases, blood chemistries, and coagulation factors remained unchanged during and after these massive transfusions. Blood transfusion at rapid rates required during pediatric liver resection can be accomplished safely if the storage lesion of the bank blood is previously corrected.     

Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug.

GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.     

H-reflex testing to determine the neural basis of movement disorders of neurologically impaired individuals.

The H-reflex is an indirect measure of the excitability levels of alpha motoneurons. The authors have developed an adaptation of H-reflex testing which allows the continuous monitoring of human alpha motoneuron activity during movement. The technique differs from previously used H-reflex testing techniques in that it utilizes a 5 Hz stimulation to elicit the H-reflex. This allows for continuous and concomitant H- and M-wave recordings during a movement. This is an improvement over past single stimulus techniques in that the time and discomfort associated with this type of experimentation is greatly decreased. The repetitive stimulation technique enables H-reflex testing to be used clinically with neurologically impaired patient populations. Thus far, the authors have used the repetitive H-reflex testing procedures to assess alpha motoneuron activation and reciprocal inhibition of antagonist musculature during voluntary movement and anticipatory postural control responses of non-disabled individuals and of individuals with cerebral palsy. This paper describes the methodology involved in this technique and reports the results obtained.