Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms

It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Infant loss during and after male replacement in gibbons

According to the sexual selection hypothesis, infanticide during resident male replacement is an adaptive strategy that has evolved because the killing of unweaned offspring sired by previous males shortens the inter‐birth intervals of the mothers whose infants are targeted and thereby increases the reproductive fitness of the perpetrator. To test this hypothesis, we describe previously unreported cases of primary male replacement for two gibbon species (Hylobates lar and Nomascus nasutus), and review all other reported cases of primary male replacement in gibbons. Overall, infants were present in nearly half of all cases (16/33, 48%) and of the 18 infants present during replacement, 50% (N = 9) disappeared within 2 months of the event. In four of the five cases where there was sufficient demographic information to identify the likely sire of the subsequent offspring of females that lost infants, the new male was believed to be the sire. Infants were also less likely to die or disappear if the new male and original resident male were possible kin. However, there was no significant difference in the age of infants between those that died or disappeared following replacement and those that survived to weaning (p = .630). Our review of takeover‐related infant loss in gibbons confirms that periods of male instability are risky for unweaned infants and that replacing males benefit from infant loss. Nevertheless, variability in the context of infant loss and difficulties related to data collection in the field make it difficult to test competing hypotheses concerning the mechanisms and functions of infanticide in the small apes.     

The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response.

Immunization with protein-containing antigens results in two types of antigen-specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re-exposure to antigen. The issue of the inter-relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen-specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub-population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity-enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B-cell population contains few, if any, cells from the early response and is less stringently selected.     

River barriers and cryptic biodiversity in an evolutionary museum

The Riverine Barriers Hypothesis (RBH) posits that tropical rivers can be effective barriers to gene flow, based on observations that range boundaries often coincide with river barriers. Over the last 160 years, the RBH has received attention from various perspectives, with a particular focus on vertebrates in the Amazon Basin. To our knowledge, no molecular assessment of the RBH has been conducted on birds in the Afrotropics, despite its rich avifauna and many Afrotropical bird species being widely distributed across numerous watersheds and basins. Here, we provide the first genetic evidence that an Afrotropical river has served as a barrier for birds and for their lice, based on four understory bird species collected from sites north and south of the Congo River. Our results indicate near‐contemporaneous, Pleistocene lineage diversification across the Congo River in these species. Our results further indicate differing levels of genetic variation in bird lice; the extent of this variation appears linked to the life‐history of both the host and the louse. Extensive cryptic diversity likely is being harbored in Afrotropical forests, in both understory birds and their lice. Therefore, these forests may not be “museums” of old lineages. Rather, substantial evolutionary diversification may have occurred in Afrotropical forests throughout the Pleistocene, supporting the Pleistocene Forest Refuge Hypothesis. Strong genetic variation in birds and their lice within a small part of the Congo Basin forest indicates that we may have grossly underestimated diversity in the Afrotropics, making these forests home of substantial biodiversity in need of conservation.     

Regulation of Avena Fatua Seed Germination by Smoke Solutions, Gibberellin A3 and Ethylene

Dormant, intact Avena fatua L. (wild oat) seeds germinate poorly at 20 °C. Removing the hulls slightly increased germination. Treatment with smoke solutions increased the germination of both intact seeds and caryopses. Exogenous GA₃, alone or in the presence of smoke solution, increased the germination of caryopses, while ACC shows a tendency to increase germination of caryopses only when applied in combination with smoke solution. Results suggest that GA₃ and ethylene, but not smoke solutions, are involved in the regulation of α-amylase activity during germination. However, the participation of smoke solutions in the control of ACC oxidase activity cannot be excluded.     

Behavioral responses of female oriental fruit flies to the odor of papayas at three ripeness stages in a laboratory flight tunnel (Diptera: Tephritidae)

Behavioral responses of adult female oriental fruit flies, Dacus dorsalisHendel, to the odor of papayas from three ripeness classes were studied using a threechoice flight tunnel bioassay. Laboratoryreared flies were allowed to respond freely to any of three papaya odors (mature green, colorbreak to one-fourth ripe, and one-half to full ripe) emanating from identical (spherical) fruit models. Five behaviors were measured in assessing the fly's relative attraction to the odors (number of landings), arrestment (total fly seconds on sphere), fly-fly interactions on the fruit model (maximum and modal fly density), and acceptance for oviposition (total eggs laid). Females showed no significant difference in total fly landings based on all age classes combined. Significant differences were noted among age classes. Females spent more total time on the sphere and showed a higher maximum density and modal fly density to ripe fruit than to green fruit odors. Ovipositional acceptance of fruit models based on the total number of eggs laid in a sphere was greater in response to the ripefruit odor than to the other two odor classes. Olfactorystimulated behavioral responses of females to the odor of ripe papayas were significantly different from the other ripeness classes for all behaviors at 8 days postemergence and then declined in 11-day-old flies. Behavioral responses were greater during the afternoon than in the morning. Observations of wild oriental fruit flies to papayas in the field indicated a preference for residing on riper fruit. The results of this study are discussed with regard to the role of olfactory inputs generated by the odor of ripening fruit on female attraction and oviposition behavior resulting in infestation of papayas by oriental fruit fly.     

Sensitive rapid detection method for viable bacterial cells

A rapid sensitive method for the detection of viable bacterial cells is described in which P³² as inorganic orthophosphate is used to label the cells. Factors affecting the uptake of P³² by cells as well as the sensitivity of the method have been explored with suspensions of Aerobacter aerogenes. The uptake of P³²O₄ is dependent on several factors. Of various incubation media tested, one composed of 0.005 m KCl, 0.002 m MgSO₄ and 10 mg/ml of glucose was found to best stimulate the uptake of the tracer. Incubation time and temperature and level of isotope and of unlabeled P also affected uptake. Labeled cells were collected on a membrane filter for measurement of radioactivity. Under optimal conditions, as few as 23 viable cells per milliliter were detected in 1 hr with 95% confidence.     

Human apolipoprotein A-I isoprotein metabolism: proapoA-I conversion to mature apoA-I

ProapoA-I (apoA-i+2 isoform) is the major apoA-I isoprotein secreted by the liver and intestine; however, it is a minor isoprotein in plasma and lymph where the major A-I apo-lipoprotein is mature apoA-I (apoA-I0, apoA-I-1, and apoA-I-2 isoforms). In the present report we provide evidence that apoA-I is rapidly and quantitatively converted to mature apoA-I, and the mature apoA-I isoforms are catabolized at equal rates. In these studies, human proapoA-I was isolated from thoracic duct chylomicrons collected during active fat absorption and mature apoA-I was isolated from plasma high density lipoproteins. The isolated lipoproteins were delipidated, fractionated by gel permeation chromatography, and the individual apoA-I isoforms were separated by preparative isoelectrofocusing. The metabolism of apoA-I isoproteins was studied in normal volunteers (N = 6) in a metabolic ward. In the first study proapoA-I and mature apoA-I (apoA-I0 isoform) were injected simultaneously into two normal subjects and the conversion of proapoA-I to mature apoA-I and the decay of radioactivity were followed in plasma and HDL over a 14-day period. ProapoA-I was rapidly and completely converted to mature apoA-I with a fractional rate of conversion of 4.0 pools/day. The average residence times of proapoA-I and mature apoA-I were 0.23 and 6.5 days, respectively. The mature apoA-I derived from proapoA-I had a residence time which was the same as the injected mature apoA-I.(ABSTRACT TRUNCATED AT 250 WORDS)