Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides.

Photosynthetic electron transfer through the cytochrome b6f complex can bypass cytochrome f.

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.

Electron transfer and stability of the cytochrome b6f complex in a small domain deletion mutant of cytochrome f.

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.

The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin.

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues. The protein is identical [as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f purified from turnip leaves. Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.

Transient acute renal failure due to nitrofurantoin poisoning.

A 3-year-old girl developed acute oliguric renal failure after accidental ingestion of nitrofurantoin (190 mg/kg body weight). The time interval between poisoning and the onset of renal failure was 9 days. She later recovered with symptomatic management.

Digital replantation. A ten-year retrospective study.

Eight hundred and two digits (592 complete amputation and 210 incomplete amputations) of 549 patients were replanted. The male to female ratio was 3:1 and the age range 1-63 years. 728 digits survived and 74 digits failed. The overall survival rate was 90.8%. Special varieties of complicated amputation were encountered in this series: (1) distal segment replantation, 139 digits, 131 digits (94%) survived; (2) digital replantation of children, 45 cases, 78 digits, average age 3.8 years, the youngest being 1 year old, 68 digits survived (88.7%); (3) rotational avulsion amputation of thumb, 26 digits, 23 digits (88.4%) survived; (4) bilateral digit amputation, 8 cases, 44 digits amputated, 38 digits replanted, including 9 digits of a ten-digit guillotined case. All the digits were salvaged. We conclude that thorough debridement, meticulous anastomosis of blood vessels and timely management of vascular crisis are keys to high survival rate, while judicious selection of indication, proper internal fixation of bone, anastomosis of as many blood vessels as possible, attentive repair of nerves and tendons, sound rehabilitation program are important measures in improving postoperative function.

Digital replantation in children.

This article reports a series of 21 cases of digital replantation in children between 14 months and 10 years of age (mean age, 3 1/2 years) who had a total of 32 digits traumatically amputated. The amputation levels were proximal phalanx in 15 digits, proximal interphalangeal joint in 3 digits, middle phalanx in 3 digits, and distal interphalangeal joint in 11 digits. All but one digit survived after replantation, for an overall survival rate of 96.6%. The longest ischemic time was 28 hours. Several technical points related to the success of this series of cases are discussed in detail.