mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.     

Prognosis of children with malignant pheochromocytoma. Report of 2 cases and review of the literature

Malignant pheochromocytomas are rare in childhood and the prognosis of children with this tumor is not well known. We present 2 pediatric observations of malignant pelvic pheochromocytoma. Symptoms in both cases were headache and hypertension. The tumor invaded the sacral bone. Angiogram helped to localize the tumor and metastases, and allowed preoperative embolization of the tumor in 1 case. The first child underwent incomplete surgical resection, (131)I-MIBG therapy and radiotherapy, and is still alive 2 years after diagnosis. The second child died from metastatic invasion a few weeks after discovery of the tumor. We reviewed previous reports of children with malignant pheochromocytomas (30 cases). Primary tumor was extraadrenal in 50% of cases. The 3-year survival rate was 73 +/- 9% (mean +/- SD). Apart from surgical resection, no particular treatment appeared to be more effective than others in reducing mortality.     

Medial plantar flap based distally on the lateral plantar artery to cover a forefoot skin defect

The authors report a simple, single-step procedure to promote the distal transfer of the instep island flap for coverage of the submetatarsal weight-bearing zone. First described in 1991 by Martin et aI, this procedure remained unknown. As opposed to the medial plantar flap, this technique proposes an instep island flap based on the lateral plantar artery. The inflow and outflow of blood is assured by the anastomosis between the dorsalis pedis and lateral plantar vessels. This approach allows for the transfer of similar tissue and provides adequate coverage of the weight-bearing zone of the distal forefoot.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide

Objective

Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.

Methods

Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca²⁺-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca²⁺ and TPP⁺ ion-selective electrodes.

Results

Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca²⁺ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca²⁺-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca²⁺/mg protein in SSM and IFM, respectively), suggestive of increased Ca²⁺ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca²⁺ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).

Conclusion

These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca²⁺-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.