Amplified cellular oncogenes in neoplasms of the human central nervous system.

Significant advances have recently been made in a number of areas concerning central nervous system (CNS) neoplasia. Particularly salient are the following: (1) gene amplification is related to increasing grade of human glioma malignancy and occurs in approximately 40% of the most common and most malignant variety of glioma, glioblastoma multiforme (GBM), (2) by far the most commonly amplified gene in glioblastomas is the epidermal growth factor receptor (EGFR) gene, which is amplified in about one third of GBMs, (3) a small percentage of GBMs amplify N-myc or the novel sequence gli, (4) the EGFR gene is rearranged in at least half of gliomas in which it is amplified, and (5) EGFR gene rearrangement results in external domain deletions that yield truncated EGF receptors. Antibodies specific for the mutant EGF receptor fusion junction have been successfully produced and provide stimulating new potential avenues for tumor imaging and therapy. For pediatric CNS neoplasms, only medulloblastoma has been investigated in adequate numbers; a small percentage exhibit amplification of either the N-myc or c-myc genes.     

Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463.

A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Br?nnstr?m et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.     

Two Types of Genetic Interaction Implicate the Whirligig Gene of Drosophila Melanogaster in Microtubule Organization in the Flagellar Axoneme

The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.     

Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes,granulocytes, lymphocytes,and platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10⁷ cells, granulocytes have 17–201 units/10⁷ cells, lymphocytes have 91–462 units/10⁷ cells, and platelets have 1.1–7.1 units/10⁷ platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.This work was supported by funds allocated by the Agricultural Experiment Station, Michigan State University. Michigan Agricultural Experiment Station Journal No. 8727.     

The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity.

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.     

Extradural haematoma: effect of delayed treatment.

The case records of patients with extradural haematomas treated in the Lothian region during 1951-60 and 1968-77 were analysed to assess the effect of delay in treatment on morbidity and mortality. Delay was defined as the time from deterioration in level of consciousness to surgical evacuation of clot. There were 83 supratentorial extradural haematomas unassociated with intradural clot or contusion. The mean delay in patients who died was 15.7 hours, while in good-quality survivors the mean delay was 1.9 hours. Mortality decreased from 33.3% during 1951-60 to 8.9% during 1968-77. In addition, good recovery without morbidity occurred in 40.7% of patients in the earlier period and 67.9% in the later period. Mean delays from deterioration in level of consciousness to operation were 9.8 and 2.4 hours in the earlier and later periods respectively. The results emphasise the need for immediate operation in patients deteriorating with extradural haematomas. Direct admission of all head-injured patients to a head and spinal injuries unit staffed by neurosurgeons resulted in minimal delay times as well as a reduction in morbidity and mortality.     

Prolyl hydroxylase activity in normal and diseased human liver.

Prolyl hydroxylase activity was determined in liver biopsy samples obtained from 10 patients. The liver prolyl hydroxylase values in patients with active hepatitis distribute into two numerical populations based on the extent of elevation over control. The first of these groups includes those with enzyme levels elevated approximately 2.5-fold over normal. Included in this group are patients with active (but nonagrressive) hepatitis and patients where advanced portal fibrosis is already established. The second group where prolyl hydroxylase is elevated approximately nine-fold is comprised of two patients with advanced clinical symptoms of active alcoholic hepatitis with evidence of aggressive cirrhosis but with only early minimal evidence of existing fibrosis.     

Conformational studies of peptides. The crystal structures of N-acetyl-L-prolinamide and N-acetyl-(S)-thiazolidine-4-carboxamide

The crystal structure of N′-acetyl-L -prolinamide and its isomorphous alalog N′-acetyl-(S)-thiazolidine-4-carboxamide was determined using highly accurate parameters obtained by room- and low-temperature data-collecting systems. Both crystals are orthorhombic, space group P2₁2₁2₁, with four molecules per unit cell held together by a hydrogen-bond system that extends in all directions. Both molecules exhibit the following conformational features: the acetyl group is in the trans configuration in respect to the tertiary amide. The primary amide is almost at right angles with respect to the mean plane of the ring and the –NH₂ group is over the ring. The pyrrolidine and thiazolidine rings were found to be rather flexible with C^β puckering in the former and sulfur in the latter.