Shiva-1: in vitro and in vivo tests of the effects of a novel,synthetic, lytic peptide on ocular cells

An investigation was undertaken to determine the toxicity of an intravitreal injection of a novel peptide drug, Shiva-1, in rabbits. The drug, a synthetic peptide modeled after lytic peptides secreted by certain insects, has antiproliferative and antibacterial properties. Initial in vitro experiments showed that the drug, at a concentration of 100 M, was toxic to both Y-79 retinoblastoma cells and human retinal pigment epithelial cells. A wide range of doses (6–1200 g) was injected into the rabbit vitreous in an attempt to determine the maximum tolerated dose. Retinal toxicity was evaluated clinically, by electroretinography, and by light microscopy. Some localized toxicity was evident at 200 g; all doses of 240 g and above were toxic. While the drug appears to exhibit a narrow range between effective and toxic doses, the results suggest that this and other peptides of similar design merit further investigation for the treatment of proliferative and infectious diseases of the eye.Supported in part by U.S. Public Health Service grants EY07541 and EY02377 from the National Eye Institute, the National Institutes of Health Services, Bethesda, MD, USA     

Genetic evolution of breast cancers III: Age-dependent variations in the correlations between biological indicators of prognosis

The influence of age on the occurrence of phenotypic features of prognostic significance was studied in relation to the DNA index values, measured on DNA histograms from a series of 1019 breast cancer patients. Globally, the distributions of all parameters showed variations with age, the most prominent being the decreases in the percentage of estrogen receptor-negative and high proliferative activity cases with increasing age. When analyzed according to the DNA index classes, all parameters were found to some extent linked with the stage of genetic evolution. However, the associations varied with age, defining two extreme groups. The younger patients (less than 40 years) presented a more complete acquisition of the aggressive phenotype and near-triploid tumors from this group were very frequently steroid hormone receptor-negative, high proliferation, and grade III. By contrast, near-triploid tumors in patients above 65 presented relatively frequently as receptor-positive, low proliferative activity, and even grade I. The correlation of the proliferative status with steroid hormone receptor content led to similar conclusions, high proliferation being more strongly correlated with the absence of estrogen and progesterone receptors in younger patients. Interestingly, the association between high proliferation and negative progesterone receptors was much weaker in patients above 55. Our results suggest that the currently established biological prognostic factors, including DNA profile, steroid hormone receptors, and histopathological grade, show patterns of association which vary with age. Of these, only progesterone receptor could be influenced by menopausal status. These findings have to be taken into consideration for future prognostic factor-related treatment decisions, but also for future methodological improvements of multivariate survival analyses.     

Homosexuality,type 1: An Xq28 phenomenon

Despite the absence of phenotypic manifestations in alternating generations characteristic of X-linked disorders, a thesis is presented that a major type of Kinsey grades 5 and 6 male homosexuality is determined by a gene in the Xq28 region. A total of 133 families in 78 kinships of male and female homosexual probands, in addition to 116 families (including those of 40 famous homosexuals) from the literature, revealed an unbalanced secondary sex ratio in the maternal generation of male, but not of female, homosexuals. On the maternal side, in this study, the ratio of all uncles to all aunts of 90 males homosexuals was 132/209, ² = 8.52, p = 0.004. On the maternal side for the total of all sources, the ratio of uncles to aunts of male homosexuals was 241/367, ² = 13.20; p < 0.0001. The male/female ratio of the total number of maternal sibships bearing homosexuals (310/628: 0.491) was a measure of fetal wastage of the mothers' male sibs: 49%. This ratio was very close to that of the total number of children born to fathers affected with any one of nine Xq28-linked male semilethal conditions (255/508: ratio 0.556); for the difference between the two populations ² = 0.859, p = 0.354. The male/female ratio of the total number of children born to female carriers of any one of these same conditions (1,232/1,062: ratio 1.16), ² = 13.8 p 0.0001, is close to that of the total number of children in homosexual sibships: 511/413, ² = 10.4, p = 0.005. Between the number of children born to Xq28 mothers and to those born of mothers of homosexuals ² = 0.581, p = 0.446. One may readily surmise that the maternal influence so often related to homosexuality may lie in the mother being a genetic carrier, with traits thereto associated. In this study, 65% of the mothers of homosexuals had no or only one live-born brother. Additional support for a genetic hypothesis is found in the occurrence of multiple instances—almost exclusively among maternal relatives—of infertility, spontaneous abortions, miscarriages, stillbirths, remaining single past age 30, and suicide. Of 109 male and 43 female homosexual index cases in the present series there were 6 instances of brother/sister homosexual sibships. Instances of homosexual parent-to-homosexual child transmission occurred as follows: one father-to-son; one father-to-daughter; one bisexual father-son; one father/mother-to-2 sons; one of mother-to-son, and one of father-to-son and father-to-bisexual daughter. There were 16 instances of presumptive transmissions from heterosexual father-to-homosexual son and 5 of heterosexual father-to-homosexual daughter. A hypothesis is proposed: Homosexuality is due to a gene at Xq28 characterized by (i) elongated cytosine-containing trinucleotide repeats upstream to translation of a gene, (ii) elongated CpG islands upstream of the trinucleotides, and (iii) cytosine methylation of CpG islands and of the cytosine-containing trinucleotides.A limited number of long tables of data, and pedigree charts, which provide the details from which this paper was developed, may be obtained on paper or on disc from the author or, for a modest fee, from the Librarian, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794.     

Mobilization of intracellular calcium stores participates in the rise of [Ca2+]i and the toxic actions of the HIV coat protein GP120

The HIV envelope glycoprotein, GP120, increases intracellular Ca2+ concentration and induces degeneration of human and animal neurons in culture. Using patch-clamp recordings and Ca2+ imaging techniques, we have now examined the contribution of intracellular stores of calcium in the effects of GP120. We report that in rat hippocampal neuronal cultures, GP120 induces a dramatic and persistent increase in [Ca2+]i which is prevented by drugs that either deplete (caffeine, carbachol, thapsigargin) or block (dantrolene) Ca2+ release from intracellular stores. In contrast, N-methyl-d-aspartate (NMDA) receptors or voltage-dependent calcium channels do not participate in these effects, as: (i) the increase in [Ca2+]i was not affected by NMDA receptor antagonists or calcium channel blockers; and (ii) and GP120 did not generate any current in whole-cell recording. Dantrolene, a ryanodine stores inhibitor, also prevented neuronal death induced by GP120. Our results show that the GP120-induced rise in [Ca2+]i originates from intracellular calcium stores, and suggest that intracellular stores of calcium may play a determinant role in the pathological actions of GP120.     

The amyloid precursor protein of Alzheimer's disease and the Abeta peptide

Alzheimer's disease is characterized by the accumulation of beta amyloid peptides in plaques and vessel walls and by the intraneuronal accumulation of paired helical filaments composed of hyperphosphorylated tau. In this review, we concentrate on the biology of amyloid precursor protein, and on the central role of amyloid in the pathogenesis of Alzheimer's disease. Amyloid precursor protein (APP) is part of a super-family of transmembrane and secreted proteins. It appears to have a number of roles, including regulation of haemostasis and mediation of neuroprotection. APP also has potentially important metal and heparin-binding properties, and the current challenge is to synthesize all these varied activities into a coherent view of its function. Cleavage of amyloid precursor protein by beta-and gamma-secretases results in the generation of the Abeta (betaA4) peptide, whereas alpha-secretase cleaves within the Abeta sequence and prevents formation from APP. Recent findings indicate that the site of gamma-secretase cleavage is critical to the development of amyloid deposits; Abeta1-42 is much more amyloidogenic than Abeta1-40. Abeta1-42 formation is favoured by mutations in the two presenilin genes (PS1 and PS2), and by the commonest amyloid precursor protein mutations. Transgenic mouse models of Alzheimer's disease incorporating various mutations in the presenilin gene now exist, and have shown amyloid accumulation and cognitive impairment. Neurofibrillary tangles have not been reproduced in these models, however. While aggregated Abeta is neurotoxic, perhaps via an oxidative mechanism, the relationship between such toxicity and neurofibrillary tangle formation remains a subject of ongoing research.     

The use of genetic instability as a clinical tool for cancer diagnosis

Human tumors exhibit two fundamentally important characteristics, extensive genetic alteration and clonality. Although it is still unclear to what extent tumors have an elevated mutational burden as compared with normal tissue, their clonality results in their ready detection. Thus, assaying tissues for clonal alterations at frequently mutated microsatellite loci represents a viable approach to cancer diagnosis. The most remarkable extension of this concept is that not only can cancer cells be detected in biological samples, but tumor DNA can also be directly detected in the serum or plasma of patients with some forms of cancer. This recent finding is currently being explored but may represent an important contribution to future diagnostic strategies.     

Diffusion of information about genetic risk within families

Recent developments in genetics are likely to exacerbate the ethical issues in clinical practice, especially with regard to privacy and disclosure of genetic information. To evaluate the behaviour of patients with respect to transmitting carrier information, we undertook a survey of 283 families with a balanced chromosomal rearrangement as a model. In these families, 1816 relatives were considered at risk and 806 of them were karyotyped (44.4%). The percentage of karyotypes performed is significantly related to the number of living children of the index couple, the reason for referral, the nature of the anomaly, the training of the counsellor and the age of the index case. This study shows the limits of the screening of at risk individuals within families, based on the willingness of the patients, and addresses practical and ethical issues around family disclosure in medical genetics.     

Cerebral energy metabolism in patients with brain lesions at normo- and hyperbaric oxygen pressures

Summary The object of this study was to ascertain the oxygen tolerance limit and the oxygenation state of the injured brain in man. While breathing air, oxygen and hyperbaric oxygen at pressures of 1.5 and 2.0 atmospheres absolute (ATA), the cerebral arteriovenous differences (AVD) for O₂, glucose, lactate, pyruvate and blood gas pressures and pH values were measured. The balance of the cerebral glucose metabolism was calculated. The results showed that the injured brain did not tolerate the exposure to an oxygen pressure of 2.0 ATA for 10 to 15 min, but exposure to 1.5 ATA for 35–40 min was tolerated and had a favorable effect on the glucose or energy metabolism of the brain as well as on the clinical course. There was a distinctly increased cerebral glycolysis while breathing air indicating insufficient oxygen delivery to the brain. The change from breathing air to oxygen resulted in a distinct inhibition of cerebral glycolysis, which indicated improved cerebral oxygenation and energy production and gave evidence for a Pasteur effect regulating the glucose metabolism of the injured brain in man. At an inspiratory oxygen pressure of 1.5 ATA we had a nearly balanced cerebral glucose metabolism indicating an adequate cerebral oxygenation and energy formation. Further increase in inspiratory oxygen pressure to 2.0 ATA (performed only in group A) increased cerebral glycolysis considerably. This was assumed to be due to cerebral oxygen poisoning resulting in disturbed oxidative energy formation. Following this alteration an extreme reduction of the cerebral glucose uptake appeared, probably due to a disturbance of the specific glucose transport system. These metabolic alterations were not accompanied by seizures or any other clinical neurological manifestation. In group B, exposed to 1.5 ATA, such alterations of the cerebral glucose metabolism did not appear. A nearly balanced cerebral glucose metabolism was found at inspiratory oxygen pressures of 1.0 and particularly of 1.5 ATA, indicating an improved oxygenation and energy production of the affected brain. Finally, a renewed increase of the cerebral glycolysis occurred following the change from breathing oxygen to air. This again indicated an insufficient oxygen delivery to the affected brain.

---

Zusammenfassung Ziel dieser Arbeit war es, die Wirkung verschiedener inspiratorischer O₂-Drucke auf den zerebralen Glukose- bzw. Energiestoffwechsel zu untersuchen. Dabei sollte insbesondere die Sauerstoff-Toleranzgrenze und der Zustand der Oxygenierung des geschädigten Hirns bestimmt werden. Unter Luft-, Sauerstoff und hyperbarer Sauerstoffatmung, d. h. bei Drucken von 1,5 und 2,0 Atmosphären, wurden die arterio-hirnvenösen Differenzen (AVD) für O₂, Glukose, Laktat, Pyruvat sowie die Blutgasdrucke und die pH-Werte gemessen. Die Bilanz des zerebralen Glukosestoffwechsels wurde bestimmt. Die Ergebnisse zeigten vor allem, daß das geschädigte Hirn eine Sauerstoffbelastung von 2,0 Atmosphären mit einer Expositionszeit von 10 bis 15 min nicht toleriert. Dagegen wurde eine Sauerstoffbelastung von 1,5 Atmosphären mit einer Expositionszeit von 35–40 min vertragen und hatte einen günstigen Einfluß auf den zerebralen Glukose- bzw. Energiestoffwechsel sowie auf den Krankheitsverlauf von traumatischen oder ischämischen Hirngewebsveränderungen. Während der Luftatmung fand sich eine erhebliche Steigerung der zerebralen Glykolyse, was auf eine mangelhafte O₂-Versorgung des Hirngewebes hinwies. Der Wechsel von Luft- auf Sauerstoffatmung führte zu einer deutlichen Hemmung der zerebralen Glykolyse. Dies zeigte eine Besserung der zerebralen Sauerstoffversorgung und Energieproduktion an und wies auf einen Pasteur Effekt bei der Regulation des Glukosestoffwechsels des geschädigten Hirns hin. Bei einem inspiratorischen Sauerstoffdruck von 1,5 Atmosphären war eine praktisch ausgeglichene Bilanz des zerebralen Glukosestoffwechsels nachweisbar, was für eine ausreichende Sauerstoffversorgung und Energiebildung des Hirns sprach. Der weitere Anstieg des inspiratorischen Sauerstoffdruckes auf 2,0 Atmosphären, der nur in Gruppe A durchgeführt wurde, bewirkte jedoch eine erhebliche Steigerung der zerebralen Glykolyse. Es ist anzunehmen, daß diese Stoffwechseländerung durch eine zerebrale Sauerstoffvergiftung hervorgerufen wurde, die vor allem zu einer Störung der oxydativen Energiegewinnung führte. Anschließend trat eine extreme Reduzierung der zerebralen Glukoseaufnahme auf, die am ehesten durch eine Störung des spezifischen Glukosetransportsystems des Hirns bedingt war. Diese Stoffwechselstörungen gingen nicht mit epileptischen Anfällen oder sonstigen klinisch-neurologischen Veränderungen einher. Bei den Patienten der Gruppe B, die nur mit einem Sauerstoffdruck von 1,5 Atmosphären belastet wurden, traten derartige Veränderungen des zerebralen Glukosestoffwechsels nicht auf. Eine praktisch ausgeglichene Bilanz des zerebralen Glukosestoffwechsels wurde bei inspiratorischen Sauerstoffdrucken von 1,0 und vor allem von 1,5 Atmosphären nachgewiesen und zeigte eine Besserung der Sauerstoffversorgung und Energiebildung des geschädigten Hirns an. Schließlich beobachteten wir nach dem Wechsel von Sauerstoff- auf Luftatmung einen erneuten Anstieg der zerebralen Glykose, was wiederum auf eine insuffiziente Sauerstoffversorgung des Hirns hinwies.

     

Reduced platelet MAO activity in healthy male students with blood group O

The association between the two genetic markers of affective disorders, ABO blood group system and platelet MAO (monoamine oxidase) activity was studied in 70 healthy young males. The platelet MAO activity of subjects with blood type O was significantly lower than that of subjects with blood type A and with blood types A + B AB + B together. This finding could constitute a "bridge" between the two genetic approaches to affective disorders.     

Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 g/kg body wt; maintenance dose: 5 g/kg body wt, once weekly) and TCDD-75 (initial dose: 75 g/kg body wt; maintenance dose: 15 g/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25=5.1%, controls=2.6%), and incompletely ossified ossa zygomatica (TCDD-25=1.8%, controls=0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25=1.3%, controls=0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls=0.6%). There was no difference in postnatal mortality (TCDD-25=1.3%, controls=1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.