Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

Studies on a Possible Functional Coupling Between Presynaptic Acetylcholinesterase and High-Affinity Choline Uptake in the Rat Brain

The relationships between presynaptic acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 microM [N-methyl-3H]ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was 1/25 of the amount passing through the synaptosomal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.     

Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.     

Presynaptic regulation of acetylcholine release in the CNS

The release of ACh appears to be under the control of autoreceptors localized on cholinergic nerve terminals. Moreover, the process can be regulated by transmitters other than ACh or by modulators either through receptor-mediated or carrier-mediated mechanisms. In this chapter we report on our recent results concerning the regulation of the release of ACh by ACh itself, 5-HT and GABA in the rat hippocampus. In particular it will be shown: 1) that the release of the cholinergic transmitter can be inhibited through muscarinic receptors of the M3 subtype; 2) that 5-HT can interact with ACh by depressing ACh release through the activation of receptors of the 5-HT1B subtype; 3) that the release of ACh can be enhanced by GABA by a novel mechanism involving a selective penetration of the amino acid into the cholinergic terminals.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.     

A refined restriction map of YAC clones spanning the entire human dystrophin gene

The enormous size of the human dystrophin gene (2300 kb) has so far hindered the analysis of its organization and the characterization at the genomic level of the deletion and duplication mutations causing Duchenne or Becker muscular dystrophy. A detailed physical map of the gene locus would considerably simplify these studies. We constructed a refined, long-range restriction map of the entire human dystrophin gene, using 12 overlapping YAC clones as DNA sources. The sites for six rare cutting enzymes (SfiI, NruI, EagI, BssHII, SacII, and NotI) were mapped by partial digest analysis of YACs over a region of 2600 kb, within a level of resolution of about 10 kb. Such a map provides the first detailed representation of the physical structure of the dystrophin gene. It will be useful for mapping unlocalized exons and, eventually, for the characterization of deletions and duplications leading to disease.     

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.