DISTANCE DATA REVISITED

Abstract— Objections to my earlier demonstration, that the branch lengths of trees fitted to distance matrices have no physical interpretation, are shown to be ill-founded. In particular the contention of Felsenstein, that fitted lengths estimate expectations of amounts of change, is shown to lead to a paradox. A method is introduced for constructing multiple trees of optimal or near-optimal fit to distance data, and this is found to give better performance than previous methods. Most published trees based on distances have been poorly chosen. Consensus trees of several trees with near-optimal fit are found to be quite poorly resolved, and it appears that molecular distances seldom provide much useful information on phylogenetic relationships.     

Evidence for essential primary amino groups in a bacterial coupling factor F1ATPase

We have found that the binding of pyridoxal-5′-phosphate to 6 primary amino groups leads to the inactivation of the enzyme. A preferential reaction of pyridoxal-5′-phosphate with the α-subunits of this enzyme can be demonstrated. The reactivity of the amino groups is influenced by various effectors. In the presence of ATP the inhibition of the ATPase activity is noncompetitive.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.     

Antibodies to the hepatitis A virus in the healthy population of Moscow

Serum specimens collected from 1002 persons in Moscow were tested for the presence of antibodies to hepatitis A virus (anti-HAV antibodies) by solid-phase enzyme immunoassay. The prevalence of these antibodies increased progressively with age from 10% in children aged 5-9 years to over 90% in the age groups of 40-49 years and over, the 50% immunity level being established at the age of 18 years. 79% of infants under 1 year were found to be immune, which was obviously due to the placental transfer of antibodies from mother to child. In a considerable part of seropositive persons over 30 years high or medium antibody titers were detected. These age groups showed a stable proportion of the low, medium and high level of anti-HAV antibodies. The prevalence of such antibodies was not related to sex. The presence of an ample amount of anti-HAV antibodies was determined in all of 18 tested lots of commercial serum immunoglobulin obtained from 3 different manufacturers.