Lack of evidence from studies of soluble protein fragments that Knops blood group polymorphisms in complement receptor-type 1 are driven by malaria

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC^a/b may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15–25 of its ectodomain. These eleven modules encompass a region (CCPs 15–17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24–25. All four CR1 15–25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15–25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K _D = 0.8–1.1 µM) and C4b (K _D = 5.0–5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.     

Melatonin in rat milk and the likelihood of its role in postnatal maternal entrainment of rhythms

The rhythm of melatonin in rat milk and the capacity of pups to synthesize and metabolize melatonin were studied. Melatonin was undetectable in milk in the light (< 21 pM), but increased rapidly 2-4 h after dark to peak at 357 +/- 66 pM at mid-dark. Oral or subcutaneous administration of melatonin to 5- and 10-day-old pups resulted in peak plasma melatonin levels 30 min after administration and rapid metabolism. Increases in pineal and plasma melatonin levels at night were detected at 5 and 6 days of age, respectively. Isoproterenol administration (2 microg/g body wt) at mid-light to day 10 pups increased plasma melatonin from 312 +/- 40 pM to 1,298 +/- 160 pM, whereas propranolol (2 microg/g body wt) suppressed nocturnal melatonin secretion from 1,270 +/- 128 pM to 395 +/- 66 pM. The rise of pineal and plasma melatonin in day 10 pups occurred 1 and 2 h after dark onset, respectively, preceding the onset in dams by 3 and 4 h, respectively. Propranolol administration to 2- and 5-day lactating dams inhibited plasma and milk melatonin at night but had no effect on their suckling pups. Transfer of melatonin via the milk is unlikely to provide an entraining signal for rat pups.     

Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood–brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited ¹²⁵I-NPS binding in the CNS when administered to rats.     

The significance of p16INK4a in cell defenses against transformation

Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.     

Pineal cells enhance choline acetyltransferase activity in sympathetic neurons

Cells derived from the neonatal rat pineal gland were cocultured with cells derived from neonatal rat superior cervical ganglia (SCG) in an attempt to determine whether a sympathetic target organ with only adrenergic properties could enhance the development of adrenergic transmitter properties in sympathetic neurons in tissue culture. Choline acetyltransferase was measured as an index of cholinergic differentiation, and tyrosine hydroxylase was measured as an index of adrenergic differentiation. As indices of total cell number and cellular volume, DNA and protein, respectively, were also measured. We found that the pineal-SCG cocultures contained ten times greater choline acetyltransferase activity than sister neuronal cultures cultured without pineal cells, thus indicating that the pineal cells enhanced cholinergic properties in the sympathetic neurons. This cholinergic enhancement was dependent upon the presence of nerve growth factor and could not be obtained with pineal-conditioned medium. Tyrosine hydroxylase activity, measured on cultures sister to those mentioned above, was low in all cultures and decreased somewhat in SCGs cultured alone. TH activity in the pineal-SCG cocultures, however, increased slightly. Some tyrosine hydroxylating activity developed in pineals cultured alone, however, and may have been responsible for the small increase in tyrosine hydroxylase activity noted in the pineal-SCG cocultures. The implications of these results for a determination of the role that target organ plays in the development of the transmitter properties of sympathetic neurons are discussed.     

Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA

The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.