Localization of melanin synthesis within the pigment cell: Determination by a combination of electron microscopic autoradiography and topographic planimetry

Summary Localization of melanin synthesis within the pigment cells of the Cloudman S-91 mouse melanoma was determined by means of a combination of high resolution autoradiography and topographic planimetry. Initial melanin biosynthesis occurred predominantly in the endoplasmic reticulum and associated ribonucleoprotein particles of the melanocytes. By measuring a number of cell organelles and employing the index of relative specific localization it could be shown that the nucleus and mitochondria are of little or no importance in the process of melanogenesis.This investigation has been supported by the following research grants: CA 06548 CB, NIH, PHS and an Institutional Grant from the American Cancer Society (to H. M. H.); CA-05887, NIH, PHS (to A. S. Z.); M-00388 and NB-00782, NIH, PHS (to J. F. H.). One of us (H. M. H.) holds a Research Cancer Development Award (5-K 3-GM-2634) of the National Institute of General Medical Sciences, Public Health Service.We are grateful to Mr. Ronald Abler for his help with the topographic measurements; to Dr. Erhard Haus for help and advice; to Mr. J. Thornby and Mr. A. P. Basu for assistance with the statistical aspects of this study; and to Mrs. Lenore Mottaz, Miss Bernice Uittenbogaard, and Mrs. Judith Strong for careful technical assistance.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

Photoperiodic influences on sexual behavior in male Syrian hamsters

The effect of photoperiodic conditions on sexual behavior was investigated in male Syrian hamsters that were either gonadally intact, or castrated and treated with low doses of testosterone throughout the experiment. Hamsters were exposed to long (LD 16:8) or short (LD 8:16) days for 7 weeks; for the next 8 weeks, either they were exposed to an intermediate daylength (LD 12:12), or daylength conditions remained unchanged. Sexual behavior was affected by photoperiod conditions in both gonadally intact animals and testosterone-treated castrates, but to different degrees. Intact males exposed to short days for 15 weeks exhibited gonadal regression, and their copulatory performance was impaired. The percentage of animals that intromitted or ejaculated was significantly reduced. Additional measures of sexual performance among the copulating males were also affected. In contrast, among the castrates with testosterone clamped at low but stable levels, the proportion of males that mounted, intromitted, or ejaculated was not affected by photoperiod. However, among the males that continued to copulate, sexual performance changes were present in the short-day castrates that resembled those displayed by the intact males. We infer that these behavioral effects in both hormonal conditions reflect primarily a difficulty in the attainment of intromission. Gonadal regression alone cannot easily account for the behavioral deficits of the intact males, because circulating testosterone levels at the end of the experiment were not significantly different between the gonadally intact hamsters and the castrated, testosterone-treated hamsters exposed continuously to short days. Males transferred from either long or short days to the intermediate-daylength condition responded behaviorally to this photoperiod as if it were a short day, that is, their ejaculatory frequency declined. We conclude that male hamsters exposed to photoinhibitory daylengths exhibit deficits in their sexual behavior, not only because endogenous levels of testosterone decrease, but also because the substrates on which this hormone acts become less responsive. We hypothesize that under physiological conditions, the episodic secretion of testosterone imposes constraints on the maintenance or restoration of copulation, and that the potent behavioral effects achieved by constant-release implants of testosterone may mask the presence of photoperiodically induced alterations in the hamster's sensitivity to this gonadal hormone.     

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine- and 1-Methyl-4-(2''-Ethylphenyl)-1,2,3,6-Tetrahydropyridine-Induced Toxicity in PC 12 Cells: Role of Monoamine Oxidase A

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), and their corresponding pyridinium species was studied in the rat pheochromocytoma PC12 cell line. MPTP and its analogues are known to be metabolized by monoamine oxidase (MAO) to dihydropyridinium intermediates which are further transformed, either enzymatically or spontaneously, into pyridinium species. MAO activity in PC12 cells is almost exclusively of the A form, and 2'Et-MPTP is a good substrate for both MAO-A and MAO-B. In contrast, MPTP is a poor substrate for MAO-A, but a good substrate for MAO-B. 2'Et-MPTP caused considerably more cell death than MPTP in the PC12 cells. However, 1-methyl-4-(2'-ethylphenyl)pyridinium and 1-methyl-4-phenylpyridinium, the corresponding pyridinium species formed from 2'Et-MPTP and MPTP, respectively, were equipotent as toxins. The toxic effects of the tetrahydropyridines and their corresponding pyridiniums were both concentration- and time-dependent. Measurements of the levels of the pyridinium species formed and the remaining tetrahydropyridine in the media indicated that 2'Et-MPTP was converted about five to seven times more readily into its toxic pyridinium species than was MPTP. There was, moreover, an excellent correlation between amount of pyridinium formed and cell death. There was also a parallel between the capacity of clorgyline and pargyline, irreversible MAO inhibitors, to decrease the formation of the pyridinium species and their capacity to protect against the toxic actions of the tetrahydropyridines. These data are consistent with the concept that the MAO-A-dependent formation of the pyridinium species from the tetrahydropyridine is a prerequisite for toxicity in PC12 cells.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an M_r of 56 668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tic revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited an M_r of approx. 34000.     

Crystallization of the aspartylprotease from the human immunodeficiency virus, HIV-1

The aspartylprotease of the human immunodeficiency virus HIV-1 (NY5) has been crystallized in a form suitable for x-ray diffraction analysis. The crystals are tetragonal bipyramids and produce an x-ray diffraction pattern that exhibits the symmetry associated with space group P4(1)2(1)2 (or its enantiomorph, P4(3)2(1)2). The unit cell parameters are a = b = 50.3 A, c = 106.8 A, alpha = beta = gamma = 90 degrees; measurable diffraction intensities are observed to a resolution of 2.5 A. Density measurements indicate one molecule of 9,400 daltons/asymmetric unit. The symmetry of this space group could accommodate the proposed active dimer species of the protease if the 2-fold axis were coincident with one of the crystallographic 2-fold axes.     

A simple method of preparing split-thickness skin grafts for meshing

A simple and rapid technique of mounting a split-thickness skin graft on the Tanner dermacarrier is presented. Initially, petroleum jelly is applied onto the grooved surface of the dermacarrier. The skin graft is not removed from the donor site; instead, the skin graft knife is withdrawn backwards, allowing the graft to slip back, smoothly and evenly, onto the donor site through the knife. The dermacarrier, with the petroleum jelly, is laid onto the graft. The graft adheres to the dermacarrier because of the jelly. No further maneuver is necessary prior to meshing. With this technique it is impossible to apply the graft cut-side up on the recipient site.