Tyrosinase Activity (TH,DO, PAGE-Defined Isozymes) and Melanin Production in Regenerating Hairbulb Melanocytes of Lethal Yellow (Ay/a), Black (a/a), Agouti (AwJ/AwJ) and Albino (a/a/c2J/c2J) Mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in participate and soluble fractions of hairbulb melanocytes of lethal yellow (A^y/a C/C), nonagouti black (a/a C/C), and albino (a/a c^2J/c^2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, A^y/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (A^wJ/A^wJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and A^y/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and A^y/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in A^y/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Agouti Alleles Influence Thiol Concentrations in Hair Follicles and Extrafollicular Tissues of Mice (Ay/a,AwJ/AwJ,a/a)

Agouti protein (AP) expression in the wild-type agouti mouse (A^wJ/A^wJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (A^y/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of A^y/a, A^wJ/A^wJ, and a/a (black) mice, and 2) adipose and spleen tissues of A^y/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. A^y/a hair follicles and serum showed highest cysteine and lowest GSH levels. A^wJ/A^wJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed A^y/a > a/a (P > 0.01). An A^y-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.     

Influence of Background Genome on Enzymatic Characteristics of Yellow (Ay/-, Avy/-) Mice

Identification of the fundamental polypeptide difference between yellow (A(y)/-, A(vy)/-) and non-yellow mice is important for biomedical research because of the influence of the yellow genotype on normal and neoplastic growth and obesity. The complexity of the "yellow mouse syndrome" makes attainment of this objective dependent on the separation of those pleiotropic enzyme differences which are secondary, and depend on the background genome, from those which are primary, and depend primarily on the agouti locus genotype.-Four of nine hepatic enzyme activities assayed simultaneously differed between eight-week-old yellow (A(y)/-, A(vy)/-) and non-yellow (A/-, a/a) male inbred and F(1) hybrid mice. Among these four, only cytoplasmic malic enzyme activity was elevated in all yellow mice, as compared with the non-yellow sibs, regardless of background genome. Glucokinase, serine dehydratase, and tyrosine alpha-ketoglutarate transaminase activities were also changed in yellow mice, but these alterations depended on the background genome.-The ratio of malic enzyme activity to citrate-cleavage enzyme activity, possibly related to the altered fat metabolism of yellow mice, was influenced by background genome as well as by the yellow genotype.--Significant deviations of enzyme activities from mid-parent values among F(1) hybrids were associated with particular background genomes; the number of such deviations was larger among yellow mice than among non-yellows and this difference was greater among C3H F(1) hybrids than among C57BL/6 F(1) hybrids.     

Agouti-Related Maturation and Tissue Distribution of oc-Melanocyte Stimulating Hormone in Wild-Type (AwJ/AwJ) and Mutant (Ay/a,a/a) Mice

Because of ectopic overproduction of agouti protein, yellow alleles (A^y and A^vy) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of α-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A^y/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-α-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A^y/a, a/a, and white-bellied agouti (A^wJ/A^wJ) mice (Strain C57BL/6J). We found no evidence that A^y/a mice possessed an imbalance of des-, mono-, and diacetylated α-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A^wJ/A^wJ) exhibited significantly decreased (P < 0.05) tissue levels of total α-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A^y/a and a/a mice.     

Effects of diapause on lethal yellow (Ay/Ay) mouse embryos.

One of the problems in studying early acting recessive lethal genes is recognizing the homozygotes prior to their demise. Molecular probes can assist in this task, but their use generally requires removal of cells and consequent damage to the embryo, which may compromise its subsequent development. The present study was undertaken in an attempt to distinguish intact, living, lethal yellow (Ay/Ay) embryos from Ay/ae and ae/ae littermates before implantation by placing them in implantation delay or diapause. After 2 days in the reproductive tract of prepubertal females, the great majority of presumptive lethal Ay/Ay embryos has failed to hatch from the zona pellucida and they exhibited a marked deficiency of cells relative to controls, particularly in the inner cell mass. This argues against a stage-specific role for the gene in implantation.     

In vitro analysis of pre- and early postimplantation development of lethal yellow (Ay/Ay) mouse embryos

Preimplantation embryos from matings between yellow heterozygous (Ay/a) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control matings (Ay/a female X a/a male, a/a female X Ay/a male, a/a female X a/a male). Most embryos were at the 8-cell stage at recovery; however fewer embryos from the experimental cross had developed to the 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P less than 0.01). The yellow (Ay/a) uterus did not contribute (P = 0.05) to delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying the class of Ay/Ay mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (10/106) of the morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (10/398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24/96) of embryos that developed successfully to the blastocyst stage failed to hatch from the zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.     

Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in regenerating hairbulb melanocytes of lethal yellow (Ay/a), black (a/a), agouti (AwJ/AwJ/) and albino (a/a/c2J/c2J) mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in particulate and soluble fractions of hairbulb melanocytes of lethal yellow (Ay/a C/C), nonagouti black (a/a C/C), and albino (a/a c2J/c2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, Ay/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (AwJ/AwJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and Ay/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and Ay/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in Ay/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

The Regulatory Role of Sulfhydryl Compounds in Melanogenesis

Since the discovery in 1967 of the phaeomelanin pathway the regulatory role of sulfhydryl compounds in epidermal melanin pigmentation has been a major focus in pigment cell research. The current state of knowledge in the field is briefly discussed, with special reference to some recent studies extending the possible targets of sulfhydryl compounds to pigment intermediates beyond the dopaquinone stage.     

In vivo development of the lethal yellow (Ay/Ay) mouse embryo at 105 hours post coitum

When histologically examined in utero at 105 hours post coitum embryos from A ^y /a × A ^y /a matings contained a mean (± SE) of 122.7 ± 6.5 nuclei per embryo; of these, 30.2 ± 2.4 nuclei were in the inner cell mass (ICM) and 92.5 ± 4.8 nuclei were within trophoblast cells. Embryos from A ^y /a × a/a matings contained a mean of 141.4 ± 10.6 nuclei per embryo of which 41.7 ± 4.5 nuclei were within the ICM and 99.7 ± 6.9 nuclei were trophoblastic. ICM cell numbers were significantly different between genotypes (P < 0.05) suggesting that homozygous A ^y allele expression selectively interferes with ICM versus trophoblast cell differentiation during preimplantation development.     

A Bacillus stearothermophilus Esterase Produced by a Recombinant Bacillus brevis Stabilized by Sulfhydryl Compounds

In the microvascular system, pericytes are located at the abdominal side of capillary endothelial cells.

To discover the role of pericytes in the microvascular system, we have analyzed the extracellular proteins secreted from pericytes isolated from microvessel fragments of rat epididymal fat pads and found that they synthesize substantial amounts of basement membrane components such as type IV collagen and laminins. Secretion of type IV collagen was markedly stimulated by ascorbic acid phosphate. Reducing and non-reducing sodium dodecyl sulfate gel electrophoresis showed that pericytes produce six laminin chains assembled into different trimeric isoforms. Two of them were similar to laminin variants produced by aortic and pulmonal endothelial cells but others were suggested to be novel variants.     

Progressive infertility in female lethal yellow mice (Ay/a; strain C57BL/6J)

Obese Ay/a females of 120 days or older, when compared to age-matched a/a controls (strain C57BL/6J), exhibited abnormal oestrous cyclicity characterized by reduced frequencies of true oestrous-stage smears, decreased mating success to proven a/a males, lowered uterine weights, and depressed ovulation rates. Exogenous gonadotrophins (PMSG/hCG) partly restored ovulation in obese Ay/a females to near control levels, demonstrating the sensitivity of Ay/a ovarian tissues to FSH and LH, at least at superovulatory levels. Concentrations of endogenous gonadotrophins and/or sensitivity of ovarian target cells to gonadotrophins may therefore be impaired in obese Ay/a females. Aberrant copulatory behaviour, reduced uterine weights, and depressed conception rates strongly suggest ovarian steroid deficiencies, perhaps secondary effects of reduced endogenous gonadotrophin activity. As in other obese rodent syndromes e.g. ob/ob, db/db, and fa/fa), a possible fundamental Ay-induced hypothalamic lesion is consistent with our data.     

Antibacterial Potential of Garlic-Derived Allicin and Its Cancellation by Sulfhydryl Compounds

Allicin (allyl 2-propenethiosulfinate), an antibacterial principle of garlic, has drawn much attention, since it has potent antimicrobial activity against a range of microorganisms, including methicillin-resistant Staphylococcus aureus. There have been many reports on the antibacterial properties of allicin, but no quantitative comparison of antibacterial activities between freshly prepared garlic extract and clinically useful antibiotics has been performed. To verify the substantial antibacterial effect of aqueous garlic extract, we compared it with those of allicin and several clinically useful antibiotics using two representative bacteria commonly found in the human environment, Gram-positive S. aureus and Gram-negative Escherichia coli. The garlic extract had more potent anti-staphylococcal activity than an equal amount of allicin. In terms of antibiotic potency against Gram-positive and Gram-negative bacteria, authentic allicin had roughly 1–2% of the potency of streptomycin (vs. S. aureus), 8% of that of vancomycin (vs. S. aureus), and only 0.2% of that of colistin (vs. E. coli). The antibacterial activity of allicin was completely abolished by cysteine, glutathione and coenzyme A, but not by non-SH-compounds. The oxygen in the structure (–S(=O)–S–) of allicin therefore functions to liberate the S-allyl moiety, which might be an offensive tool against bacteria.     

Physical and Genetic Characterization of a 75-Kilobase Deletion Associated with A(l), a Recessive Lethal Allele at the Mouse Agouti Locus

The agouti locus (A) of the mouse determines the timing and type of pigment deposition in the growing hair bulb, and several alleles at this locus are lethal when homozygous. Apparent instances of intragenic recombination and complementation between different recessive lethal alleles have suggested that the locus has a complex structure. We have begun to investigate the molecular basis of agouti gene action and recessive lethality by using a series of genetically linked DNA probes and pulsed field gel electrophoresis to detect structural alterations in radiation-induced agouti mutations. Hybridization probes from the Src and Emv-15 loci do not reveal molecular alterations in DNA corresponding to the ae, ax, and al alleles, but a probe from the parotid secretory protein gene (Psp) detects a 75-kilobase (kb) deletion in DNA containing the non-agouti lethal allele (al). The deletion is defined by a 75-kb reduction in the size of BssHII, NotI, NruI and SacII high molecular weight restriction fragments detected with the Psp probe and is located between 25 kb and 575 kb from Psp coding sequences. Because the genetic distance between A and Emv-15 is much less than A and Psp, there may be a preferred site of recombination close to Psp, or suppression of recombination between A and Emv-15. The al deletion has allowed us to determine the genotype of mice heterozygous for different recessive lethal alleles. We find that three different recessive lethal complementation groups are present at the agouti locus, two of which are contained within the al deletion.     

Quantitation of Gibberellins A(1), A(3), A(4), A(9) and a Putative A(9)-Conjugate in Grafts of Sitka Spruce (Picea sitchensis) during the Period of Shoot Elongation

The levels of endogenous gibberellin A₁ (GA₁), GA₃, GA₄, GA₉, and a cellulase hydrolyzable GA₉ conjugate in needles and shoot stems of mature grafts of Sitka spruce (Picea sitchensis [Bong.] Carr.) grown under environmental conditions that were either inductive, hot, and dry, or noninductive, cool, and wet, for flowering, were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated [²H₂]GA₁, GA₃, GA₄, and GA₉ as internal standards. The samples were taken when the shoots had elongated about 30, 70, and 95% of the final shoot length and 17 days after elongation had terminated. The concentration of putative GA₉-conjugate, estimated by GCSIM of GA₉ after cellulase hydrolysis of the highly water soluble fraction, was 33 nanograms per gram fresh weight in the needles of both heat and drought- and cool and wet-treated plants sampled just after bud burst. The concentration gradually decreased to a final value of 13 nanograms per gram fresh weight in the heat and drought-treated grafts and 6 nanograms per gram fresh weight in the cool and wet-treated grafts. The stems contained no detectable putative GA₉ conjugate. Free GA₉ was highest in heat and drought-treated material. For plants subjected to this treatment, GA₉ increased from 22 to 32 nanograms per gram fresh weight in needles and from 1 to 22 nanograms per gram fresh weight in stems during the rapid stem elongation phase. By day 17, after cessation of shoot elongation, GA₉ had decreased to 12 nanograms per gram fresh weight in needles and 9 nanograms per gram fresh weight in the shoot stems. The cool and wet-treated material also showed an increase in GA₉ concentration during shoot elongation. However, the concentration was not as high and was also delayed compared with heat and drought-treated material. By day 17, after cessation of shoot elongation, GA₉ concentration was 9 nanograms per gram fresh weight in needles and 5 nanograms per gram fresh weight in stems for cool and wet treatment plants. The concentration of GA₄ was very low in tissue from both treatments. Fluctuation in concentration of the more polar gibberellins, GA₁ and GA₃, showed the same pattern as fluctuations in the content of GA₉. However, the heat and drought-treated material had lower amounts of GA₁ and GA₃ during the later phases of shoot elongation, than the cool and wet-treated material. These results imply differential metabolism between clones treated with conditions inductive and noninductive for flowering. Higher concentrations of putative GA₉ conjugate and free GA₉ in the hot and dry treatment indicate a higher capacity of synthesizing, for flowering, the physiologically important GA₄ in the heat and drought-treated material. This synthesis does not, however, result in a buildup of the GA₄ pool, probably because of a high turnover rate of GA₄. The cool and wet-treated material had higher amounts of GA₁ and GA₃, indicating that the differentiation was preferentially directed toward vegetative growth.     

Dermal-epidermal interactions and the site of action of the yellow (Ay) and nonagouti (a) coat color genes in the mouse

The alleles at the agouti locus in mice determine whether eumelanin or pheomelanin is synthesized by the follicular melanocytes. Previous studies have indicated the dermis as the site of action of the agouti alleles, while implying that the epidermis plays only a passive role. Using methods of dermal-epidermal recombinations of embryonic yellow (A^y) and nonagouti (a) mouse skin, the study reported here indicates that the epidermis, as well as the dermis, plays a role in the action of the agouti alleles. When yellow dermis is recombined with nonagouti epidermis, the hairs produced contain only pheomelanin, thus substantiating the role of the dermis. However, the reciprocal combination of nonagouti dermis and yellow epidermis also produces hairs containing pheomelanin, indicating a more important role for the epidermis. The role of the dermal-epidermal interactions in the action of the alleles at the agouti locus is discussed.