Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction

The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.     

Specific localization of the respiratory alternative oxidase in meristematic and xylematic tissues from developing soybean roots and hypocotyls.

We used tissue printing and specific immunostaining to examine the localization of the alternative oxidase (AOX) protein in correlation with measurements of AOX capacity. Selected root and hypocotyl regions were analyzed during the first 14 d of growth. It is shown that AOX protein is localized in the apical meristem and in developing xylem. The temporal pattern of expression is coincident with the evolution of AOX capacity. Data suggest that AOX expression is linked to xylem differentiation. Since heat is a major product of the alternative pathway, we speculate that thermogenesis is implicated in morphogenesis.     

The reproductive ecology and abundance of Symptevygia bonapavtii endemic to the south-west Atlantic

Logistical ogives gave an estimated L _T50 value (i.e. the total length at which 50% individuals are sexually mature) of 635 mm for female and 650 mm for male Sympterygia bonapartii . Mature individuals of both sexes had significantly larger livers than immature ones and females had a significantly heavier liver than males. Clasper elongation was the first step in male maturation, followed by clasper calcification and the development of alar thorns. In mature females, the right ovary was larger than the left, which was apparently due to differences in stroma tissue. Gonadosomatic index and diameter of ovarian follicles of mature females peaked in late spring and was at a minimum from late summer and through the winter. Juvenile S. bonapartii were more abundant near estuarine areas during winter, and adults appeared in estuaries by late spring and summer. Females carrying egg-cases were found near the shore in late spring and egg-cases were found in benthic samples only in shallow waters suggesting that S. bonapartii deposits egg-cases in shallow waters during late spring-summer and that nursery areas are in outer estuarine zones. In the southern part (38°–42° S) of the study area S. bonapartii showed a strong movement to shallow waters in late spring and summer, spreading over the entire coastal area in winter. These movements are discussed in relation to water temperature and trophic interactions.     

Neo-adjuvant chemo-(immuno-)therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin + 5-fluorouracil (5-FU) with cisplatin + 5-FU + recombinant interleukin 2

We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical response and toxicity of the combination chemotherapy cisplatin + 5-FU with the same combination plus s.c. recombinant interleukin-2 (rIL-2) in patients with advanced (stage III–IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical Al Sarraf treatment: 100 mg/m² cisplatin i.v. on day 1 plus 1000 mg m⁻² day⁻¹ 5-FU on days 1–5 as a continuous infusion. Regimen B was the same as regimen A plus 4.5 MIU/day rIL-2 s.c. on days 8–12 and 15–19. Treatment was repeated every 3 weeks for three cycles. A total of 33 patients were enrolled in the study; 30 were evaluable for toxicity and 28 for response. Seventeen patients were assigned to group A and 16 were assigned to group B. Three patients (20%) of group A and 4 (31%) of group B had a complete response, 9 patients (60%) of group A and 6 (46%) of group B had a partial response, with an overall response rate of 12 patients (80%) for group A and 10 patients (77%) for group B. Two patients (13%) of group A and 3 patients (23%) group B had stable disease; 1 patient (7%) of group A had progressive disease. Thus, there was not a statistically significant difference in response rate between the two groups and therefore there was no benefit from the addition of immunotherapy with rIL-2 to the standard chemotherapy. Both regimens were well tolerated. There were 2 toxic deaths (6.7%), 1 from hematological causes in group A and 1 from cardiac causes in group B. Myelosuppression and gastrointestinal toxicity, mainly nausea/vomiting and stomatitis, were the most frequent toxicities. The calculated number of patients for the sample has not yet been reached; however, the projection of our present results suggests that it is highly improbable that a clinically significant difference between the two treatment groups will be observed even if the calculated patient sample size is achieved. Received: 9 April 1998 / Accepted: 30 June 1998     

Further localization of a gene for paroxysmal dystonic choreoathetosis to a 5-cM region on chromosome 2q34

Paroxysmal dystonic choreoathetosis (PDC) is a rare neurological disorder characterized by episodes of involuntary movement, involving the extremities and face, which may occur spontaneously or be precipitated by caffeine, alcohol, anxiety, and fatigue. PDC is transmitted as an autosomal dominant trait with incomplete penetrance. A gene implicated in this paroxysmal disorder has been mapped to a 10–15 cM region on chromosome 2q31–36 in two families. We describe a third family with PDC. Two-point linkage analyses with markers linked to the candidate PDC locus were performed. A maximum two-point LOD score of 4.20 at a recombination fraction of zero was obtained for marker D2S120, confirming linkage to the distal portion of chromosome 2q. The anion exchanger gene, SLC2C, maps to this region, but the family was poorly informative for polymorphic markers within and flanking this candidate gene. Haplotype analysis revealed a critical recombination event that confines the PDC gene to a 5-cM region bounded by the markers D2S164 and D2S377. We compared the haplotype in our family with that in another chromosome 2-linked PDC family, but did not detect a region of shared genotypes. However, identifying a third family whose disease maps to the same region and narrowing the critical region will facilitate identification of the 2q-linked PDC gene. Received: 10 June 1997 / Accepted: 17 September 1997     

Passive recovery of an island bird community after rodent eradication

The number and scale of island invasive species eradications is growing, but quantitative evidence of the conservation efficacy of passive recovery is limited. We compare relative abundances of breeding birds on Hawadax Island (formerly named Rat island), Aleutian Archipelago, Alaska, pre- and post- rat eradication to examine short-term (<1 year post-eradication) changes due to rodenticide application, and medium-term (5 years post-eradication) changes due to the absence of invasive rats. In the short term, Bald Eagle (Haliaeetus leucocephalus) numbers decreased from 24 individuals pre-eradication to two individuals <1 year post-eradication, but recovered to 10 individuals (42 % of pre-eradication) 5 years post-eradication, with all individuals nesting (63 % of the pre-eradication nesting). Five years post-eradication relative abundances of most terrestrial birds surveyed using point counts either significantly increased [Gray-crowned Rosy Finch (Leucosticte tephrocotis), Lapland Longspur (Calcarius lapponicus), Snow Bunting (Plectrophenax nivalis), Song Sparrow (Melospiza melodia)] or did not differ [Pacific Wren (Troglodytes troglodytes)]. Shorebirds also increased 5 years post-eradication with Black Oystercatchers (Haematopus palliates) increasing fivefold, and Rock Sandpiper (Calidris ptilocnemis) nesting increasing from one to five nests. We confirmed two species of ground nesting seabirds [Tufted Puffin (Fratercula cirrhata) and Leach’s Storm-petrel (Oceanodroma leucohoa)] as nesting (puffin) or engaged in courtship behavior (Storm-petrel) 5 years post-eradication. Our results indicate that despite the short-term impact on Bald Eagles, and without further human intervention, most terrestrial and marine birds have newly-colonized, re-colonized, or increased in abundance following the eradication of invasive rats.