Accumulation of D-aspartic acid with age in the human brain.

An age-related accumulation of D-aspartic acid was detected in the white matter of ten normal brains from individuals aged 30 to 80 years. Gray matter showed no systematic increase in D-aspartic acid. The rate constant for D-aspartate formation in the brain is equal to the predicted value calculated for 37 degrees C. Accumulation of the uncommon D-aspartate isomer in myelinated white matter implies that there is little or no turnover of this tissue, and this may have a bearing on dysfunction of the aging brain or on other diseases of myelin.

Development of a flexible and specific gene delivery system for production of murine tumor models.

To develop models of human cancer we have expressed the avian retroviral receptor, TVA, under a variety of mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with avian leukosis virus-derived gene vectors. TVA-based retroviral gene transfer offers advantages over current murine models of human cancer. A single transgenic mouse line can be used to evaluate multiple genetic lesions, individually and in combination. Furthermore, mutant genes are introduced somatically into animals, as occurs in the majority of naturally occurring tumors. Because the avian viral vectors replicate only in avian cells, the viral receptor in infected transgenic mouse cells remains available for multiple rounds of infection with different ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists.

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.

The role of D-aspartic acid and N-methyl-D-aspartic acid in the regulation of prolactin release.

In this study, using an enzymatic HPLC method in combination with D-aspartate oxidase, we show that N-methyl-D-aspartate (NMDA) is present at nanomolar levels in rat nervous system and endocrine glands as a natural compound, and it is biosynthesized in vivo and in vitro. D-aspartate (D-Asp) is its natural precursor and also occurs as an endogenous compound. Among the endocrine glands, the highest quantities of D-Asp (78 +/- 12 nmol/g) and NMDA (8.4 +/- 1.2 nmol/g) occur in the adenohypophysis, whereas the hypothalamus represents the area of the nervous system where these amino acids are most abundant (55 +/- 9 and 5.6 +/- 1.1 nmol/g for D-Asp and NMDA, respectively). When D-Asp is administered to rats by ip injection, there is a significant uptake of D-Asp into the adenohypophysis and a significant increase in the concentration of NMDA in the adenohypophysis, hypothalamus and hippocampus, suggesting that D-Asp is an endogenous precursor for NMDA biosynthesis. Experiments conducted on tissue homogenates confirm that D-Asp is the precursor of the NMDA and that the enzyme catalyzing this reaction is a methyltransferase. S-adenosyl-L-methionine (SAM) is the methyl group donor. In vivo experiments consisting of ip injections of sodium D-aspartate show that this amino acid induced a significant serum PRL elevation and this effect is dose and time dependent. In vitro experiments conducted on isolated adenohypophysis or adenohypophysis coincubated with the hypothalamus, showed that the release of PRL is caused by a direct action of D-Asp on the pituitary gland and also mediated by the indirect action of NMDA on the hypothalamus. Then, the latter induces the release of a putative factor that in turn stimulates the adenohypophysis reinforcing the PRL release. In conclusion, our data suggest that D-Asp and NMDA are present endogenously in the rat and are involved in the modulation of PRL release.

Synthesis and some biological activities of the tyrosine-8 analog of substance P.

[Tyr8]-substance P, an undecapeptide having the structure Arg-Pro-Lys-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH2, has been synthesized by the solid-phase technique on a Beckman automatic peptide synthesizer, appropriately purified and biologically characterized. At twice the dosage, [Tyr8]-substance P showed the same biological activity response as synthetic substance P for stimulation of contraction of the isolated guinea pig ileum and for decrease in the systemic blood pressure of dogs. On the dog's blood pressure, no qualitative differences were observed, but on the isolated gut, the Tyr8 analog gave a more gradual increase in the muscle tone than synthetic substance P. [Tyr8]-substance P released, in vitro, the luteinizing and follicle-stimulating hormones at a very high dosage but did not release growth hormone, prolactin, or thyrotropin.

Quinoxaline studies. 23. Potential antimalarials. Substituted 5,8-dimethoxyquinoxalines.

Two series of 2,3-disubstituted 5,8-dimethoxy-6-[N- (omega-dimethylaminoalkyl) amino] quinoxalines were prepared: the first series with identical 2,3-substituents H, CH3, C6H5, C6H4-4-Cl, and CH2C6H5; and the second with identical styryl groups CH=CHC6H5, CH=CHC6H4-4-Cl, CH=CHC6H3-3,4-C12, CH=CHC6H4-4-F, CH=CHC6H4-4-CF3, and CH=CHC6H4-4-NO2. None of the substances possessed antimalarial activity; several were toxic at highest dosage levels.

Appearance of D-amino acids during aging: D-amino acids in tumor proteins.

In 1939 Kögl and Erxleben [1-4] reported that tumor proteins contain appreciable amounts of D-amino acids, specifically glutamic acid, valine, leucine and lysine, implying that both the initiation and autonomous character of tumors depends on the formation and maintenance of these D-amino acids in the cell proteins. This postulate remained highly controversial for over 10 years, during which time several papers both supporting and refuting this hypothesis were published. The dispute existed almost entirely between Kögl, a vigorous and able protagonist at the University of Utrecht, Netherlands, and an impressive array of equally vigorous and able dissenters in the United Kingdom and Germany. An excellent review of both sides of this controversy was written by Miller in 1950 [5]. After many years and much effort the controversy then seemed to be put to rest. However, more than 40 years later the development of much more refined analytical techniques for the resolution and detection of amino acid enantiomers provided more definitive evidence that D-amino acids are not common to all tumor tissues and probably are not integral to the cancer process. This is not surprising when one considers that a tumor consists of fast-growing cells. Thus, there would not be sufficient time for any L-amino acid to racemize to the D isomer. Some D-amino acids may originate in foods consumed, but it is uncertain whether enzyme systems are able to incorporate D-amino acids into tumor proteins during growth. Nevertheless, if significant levels of D-amino acids were to be found in tumor proteins, the implications could be far-reaching. Confirmation of the presence of D-amino acids at any concentration in tumors would provide new insights into the mechanism for autogenesis and maintenance of tumors.

Presence of D-alanine in proteins of normal and Alzheimer human brain.

This report constitutes the first demonstration of the presence of D-alanine in the proteins of the human nervous system. Proteins of the frontal lobe white and gray matter of human brains, both normal and Alzheimer subjects, contain D-alanine at concentrations between 0.50 and 1.28 mumol/g of wet tissue, 50-70-times lower than the concentration of L-alanine. Both white and gray matter of Alzheimer brains contain D-alanine 1.4-times higher than the respective regions of normal brains. The gray matter proteins of Alzheimer brains show a highly significant 8% decrease in total alanine content, when compared with normal brain gray matter proteins. Since Alzheimer's disease is exhibited by deterioration of the gray matter, the occurrence of elevated D-alanine levels in the gray matter of Alzheimer brains is a significant discovery and raises the question whether this enantiomer causes the degeneration of the gray matter proteins in Alzheimer's disease, or whether it is an effect of the disease.

Quantification of D-aspartate in normal and Alzheimer brains.

Using a new procedure to hydrolyze proteins without provoking racemization of the amino acids and using enzymatic methods to determine D- and L-aspartate (Asp), we have quantified the content of protein-bound D-aspartate (both D-aspartic acid and D-asparagine) of human brain white and gray matter proteins from normal and Alzheimer subjects. The D-enantiomer is present in brain proteins at mean concentrations between 0.48 and 0.90 mumol/g of wet tissue, corresponding to concentrations 34-82 times lower than that of L-aspartate. The highest levels of D-aspartate were found in Alzheimer gray matter (0.60-0.90, mean 0.69 mumol/g of wet tissue). When expressed as the percentage of total (i.e. D- plus L-) aspartate, %D = [D/(D + L)] x 100, the Alzheimer brains show a significantly higher content of D-aspartate in both gray matter (2.08%) and white matter (1.80%) than in the corresponding tissues of normal brains (1.65% in gray, 1.58% in white).

Free D-serine concentration in normal and Alzheimer human brain.

We have analyzed both free L- and D-serine in frontal cortex of normal and Alzheimer human brain by high-performance liquid chromatography (HPLC). There was no significant difference between the two brains. In normal brain, L- and D-serine concentrations were 666 +/- 222 and 66 +/- 41 nmol/g of wet tissue, respectively, and the ratio of D-isomer to L-isomer (D/L) was 0.099 +/- 0.031. In Alzheimer brain, the concentrations were 750 +/- 150 and 66 +/- 40 nmol/g, respectively, and the D/L ratio was 0.086 +/- 0.040. Thus, it was shown that the free D-serine concentration in the Alzheimer brain was comparable to that in the normal brain.

Free D-aspartate and D-alanine in normal and Alzheimer brain.

In this report we present evidence for the presence of free D-aspartic acid (D-Asp) and D-alanine (D-Ala) in the white and gray matter of normal human brains and brains of individuals with Alzheimer's disease. D-Asp occurs at about the same concentration in the gray matter of both normal (18.6 nmol/g) and Alzheimer (14.8 nmol/g) brains, whereas in white matter its concentration is more than two times higher in normal than Alzheimer brains (22.4 and 10.5 nmol/g, respectively). D-Ala occurs in white matter at approximately the same concentration in both normal and Alzheimer brains (12.3 and 13.8 nmol/g, respectively), whereas in Alzheimer gray matter the D-Ala concentration is more than twice that found in normal gray matter (20.8 and 9.5 nmol/g, respectively). However, when the results are expressed as a percentage of D-amino acid/D+L, only small differences occur in all tissues examined.

Racemized D-aspartate in Alzheimer neurofibrillary tangles.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo posttranslational modification by racemization to D-aspartate. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a greater number of these racemized D-aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Synthetic inhibitors of carboxypeptidase N.

Further to explore the functions of carboxypeptidase N (CPN) in vivo, we undertook two studies to find CPN inhibitors of high potency and relatively long duration of action. In each study we examined for inhibition of hydrolysis of [3H]benzoyl-Ala-Arg using pure bovine serum CPN or human serum. In the first such study we synthesized a series of acyl amino acids and acyl di - and tripeptides containing arginine, lysine or both. All proved to be weak inhibitors (Ki = 10(-3) to 10(-4) M). N alpha-carbamoyl-Arg was the strongest: Ki = 3.5 X 10(-5) M. In the second study we prepared S-acyl (thio ester) derivatives of the highly potent CPN inhibitor 2-mercaptomethyl-3-guanidinoethylthiopropionic acid (2-MGP), as certain S-acyl groups markedly increase the duration of captopril, another mercapto-containing compound. Acetyl-, Boc-phenylalanyl-, phenylalanyl-, benzoyl-alanyl-, alanyl-, and Boc-alanyl-2-MGP retained the high potency of 2-MGP in vitro. Although Ala-2-MGP exerted maximum effects in vivo, like those of 2-MGP, the duration of action of Ala-2-MGP was slightly shorter than that of 2-MGP. These results indicate that the mercapto group of 2-MGP can be taken up in some forms of thioester linkage and still remain virtually the full potency of 2-MGP itself. Thus, it appears that a free mercapto function is not essential for the action of 2-MGP.

Keratoacanthoma of the head and neck.

Four representative cases of keratoacanthoma of the head and neck are presented; one of which contained squamous cell carcinoma at its base. Although keratoacanthoma has become recognized as a distinct clinical entity, its similarities to squamous cell carcinoma frequently result in difficult diagnosis. A discussion of the disease spectrum, diagnosis and management is presented. Prompt and complete surgical excision of head and neck keratoacanthoma is recommended.

Current levels of noise in an urban environment.

In 1961, the International Organization for Standardization prescribed upper tolerance limits for noise generated out-of-doors in residential districts. Between 1963 and 1970, vehicular traffic in the UK increased in volume by approximately 40%. This paper describes studies made throughout 1971 in which both peak and ambient noise-levels prevailing inside and outside buildings were measured. These levels were found greatly in excess of those regarded as tolerable ten years previously. Two methods for reducing noise-levels were considered. First, a barrier designed with the primary intention of reflecting rather than absorbing noise; secondly, a relatively simple form of double-glazing fitted to existing window frames. The barrier succeeded in reducing peak noise-levels but failed to influence ambients. The double-glazing attenuated both peak and ambient noise-levels significantly. Attention is drawn to the possibility of noise generated within buildings themselves becoming a source of discomfort for occupants and of annoyance to those outside. Noise-levels rising beyond 100 dB(A) were measured during evening business in the bar of a local hotel. A summary of data referring to noise-levels in the outdoor environment reveals that the upper tolerance limits prescribed by I S O are now being exceeded by 20 dB(A), or more, throughout 18 hours of the day. The findings are discussed in relation to the inevitable limits soon to be reached in adaptation of the human hearing mechanisms to increasing environment noise.

Recognizing human faces.

Information as to identity appears to be concentrated in and around certain areas of shapes and objects whereas other parts are more-or-less redundant. The experiments described in this paper were intended to establish which particular features of human faces, both in isolation and combination, convey most information for recognition. The results indicate the relative importance to recognition of different facial features. However, certain faces appear to embody idiosyncratic cues while some are confused consistently with others whom they appear to resemble.