Vibration as a possible explanation for putative electromagnetic field effects: a case study on marine diatoms.

PURPOSE: We examined the conjecture that mechanical vibration, being an uncontrolled and variable factor from one trial to another, might explain the inconsistency of results from investigations of the influence of extremely low frequency electromagnetic fields on the Ca-dependent motility of marine diatoms (Amphora coffeaeformis, strain #2038). MATERIALS AND METHODS: Owing to slight differences in culture of diatoms in previous studies, culture techniques are described in detail here. Diatoms showed maximal motility in logarithmic growth and motility was dependent on external [Ca], reducing at <.25 mM added Ca. Ninety-six different vertical vibration treatments were applied to the petri dishes containing the agar on which the diatoms were placed. The envelope of amplitudes varied from 50-500 pmicro at 10Hz to 100 nm-l1mmicro at 500 Hz. RESULTS: No significant effect of the mechanical vibrations on the motility response of diatoms was observed. We were unable to impose deliberate vibrations in a horizontal direction though some component of horizontal movement was probably present in our tests. CONCLUSIONS: The results imply that the variability of earlier experiments with extremely low frequency electromagnetic fields is unlikely to be ascribable to mechanical vibration.

Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line.

It was examined whether ethylene induces programmed cell death in a cell cycle-specific manner. Following synchronization of the tobacco TBY-2 cell line with aphidicolin and its subsequent removal, ethylene was injected into the head space of 300 cm(3) culture flasks at 0 h or 3.5 h later and cells were sampled for 26 h. There were significant increases in cell mortality at G(2)/M in both the 0 h and 3.5 h ethylene treatments, and for the latter treatment, another peak in S-phase. The effect at G(2)/M was greater in the 3.5 h treatment, but was ameliorated by the simultaneous addition of silver nitrate (1.2 microM). In addition, the 3.5 h ethylene treatment resulted in a 1 h delay in the characteristic rise in the mitotic index following aphidicolin-induced synchrony. The addition of silver nitrate alone (1.2 microM), also delayed the entry of cells into mitosis but had no effect on cell cycle length compared with the controls (14 h throughout all treatments) but it induced a peak of mortality 2.5 h after its addition. Nuclear shrinkage was also a characteristic feature of dying cells at G(2)/M. Using Apoptag, an in situ apoptosis detection kit, nuclear DNA fragmentation was observed in the TBY-2 cells which were often isolated on the end of a filament of normal cells. In the 3.5 h ethylene treatment, a marked increase was noted in the percentage of such cells at the G(2)/M transition compared with the controls. Hence, the data show cell death occurring at a major phase transition of the cell cycle and the observations of nuclear shrinkage, isolation of dying cells and nuclear DNA fragmentation suggest a programmed mechanism of cell death exacerbated by ethylene treatment.

Kinetic analysis of the stepwise formation of a long-range DNA interstrand cross-link by a dinuclear platinum antitumor complex: evidence for aquated intermediates and formation of both kinetically and thermodynamically controlled conformers.

Reported here is a detailed study of the kinetics and mechanism of formation of a 1,4 GG interstrand cross-link by [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(n)NH(2))](2+) (1,1/t,t (n = 6), 1), the prototype of a novel class of platinum antitumor complexes. The reaction of the self-complementary 12-mer duplex 5'-[d(ATATGTACATAT)(2)] with (15)N-1 has been studied at 298 K, pH 5.4, by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Initial electrostatic interactions with the duplex are observed for 1 and the monoaqua monochloro species (2). Aquation of 1 to yield 2 occurs with a pseudo-first-order rate constant of (4.15 +/- 0.04) x 10(-5) s(-1). 2 then undergoes monofunctional binding to the guanine N7 of the duplex to form 3 (G/Cl) with a rate constant of 0.47 +/- 0.06 M(-(1) s(-1). There is an electrostatic interaction between the unbound [PtN(3)Cl] group of 3 and the duplex, which is consistent with H-bonding interactions observed in the molecular model of the monofunctional (G/Cl) adduct. Closure of 3 to form the 1,4 GG interstrand cross-link (5) most likely proceeds via the aquated (G/H(2)O) intermediate (4) (pseudo-first-order rate constant = (3.62 +/- 0.04) x 10(-5) s(-1)) followed by closure of 4 to form 5 (rate constant = (2.7 +/- 1.5) x 10(-3) s(-1)). When closure is treated as direct from 3 (G/Cl) the rate constant is (3.39 +/- 0.04) x 10(-5) s(-1). Closure is ca. 10-55-fold faster than that found for 1,2 GG intrastrand cross-link formation by the diaqua form of cisplatin. Changes in the (1)H and (15)N shifts of the interstrand cross-link 5 indicate that the initially formed conformer (5(i)) converts irreversibly into other product conformer(s) 5(f). The NMR data for 5(i) are consistent with a molecular model of the 1,4 GG interstrand cross-link on B-form DNA, which shows that the NH(2) protons have no contacts except with solvent. The NMR data for 5(f) show several distinct NH(2) environments indicative of interactions between the NH(2) protons and the DNA. HPLC characterization of the final product showed only one major product peak that was confirmed by ESI-FTICR mass spectroscopy to be a cross-linked adduct of (15)N-1 and the duplex. The potential significance of these findings to the antitumor activity of dinuclear platinum complexes is discussed.

Steric control of stereoselective interactions between the platinum(II) complex [PtCl2(1,4-diazacycloheptane)] and DNA: comparison with cis-[PtCl2(NH3)2] and [PtCl2(ethane-1,2-diamine)] using DNA binding and molecular modeling studies.

The rate and extent of binding of [PtCl2(hpip)] (hpip=homopiperazine-1,4-diazacycloheptane) and cis-[PtCl2(NH3)2] to calf thymus DNA was measured using atomic absorption spectroscopy and it was found that [PtCl2(hpip)] bound both more rapidly and to a greater extent than did cis-[PtCl2(NH3)2]. The binding of [PtCl2(hpip)] and [PtCl2(en)] (en=ethane-1,2-diamine) to salmon sperm DNA and to synthetic, self-complementary 10-base-pair and 52-base-pair oligonucleotides was studied using enzymatic digestion and HPLC analysis of the products. [PtCl2(hpip)] forms approximately two-fold fewer GpG and ApG intrastrand adducts and concomitantly more monofunctional adducts than does [PtCl2(en)]. In the case of [PtCl2(hpip)], two GpG adducts, corresponding to the different orientations of the hpip ligand with respect to the DNA, were observed in a 1:3.3 ratio. The minor product corresponds to the orientation in which the bulkier propylene chain of the hpip ligand is adjacent to, and makes close contacts with, the floor of the major groove. When the reaction was repeated with a synthetic oligonucleotide decamer duplex, the ratio of the two forms was approximately 1:1.9 and with the 52-mer duplex it was 1:2.4, revealing an apparent systematic dependence of stereoselectivity on nucleotide size. Computer modeling of the two adducts formed by [PtCl2(hpip)] and those formed by [PtCl2(en)] and cis-[PtCl2(NH3)2] revealed that non-bonded interactions between the hpip ligand and the DNA were probably responsible for both the decreased proportion of GpG adducts formed by [PtCl2(hpip)] and the stereoselectivity exhibited in the formation of these adducts. This is the first case in which the stereoselectivity can be ascribed to steric factors alone.

Structure, stability, and interconversion barriers of the rotamers of cis-[Pt(II)Cl(2)(quinoline)2] and cis-[Pt(II)Cl(2)(3-bromoquinoline)(quinoline)] from X-ray crystallography, NMR spectroscopy and molecular mechanics evidence.

Reported are the preparations of cis-[PtCl(2)(quinoline)(2)] and cis-[PtCl(2)(3-bromoquinoline)(quinoline)] and an investigation of the stabilities and interconversion of the rotamer forms of these complexes. Both head-to-head (HTH) and head-to-tail (HTT) rotamer forms are found in the crystal structure of cis-[PtCl(2)(quinoline)(2)]. The NOESY NMR spectrum of cis-[PtCl(2)(quinoline)(2)] in dmf-d(7) at 300 K is consistent with conformational exchange brought about by rotation about the Pt-N(quinoline) bonds. H.H nonbonded distances between H atoms of the two different quinoline ligands were determined from NOESY data, and these distances are in accord with those observed in the crystal structure and derived from molecular mechanics models. cis-[PtCl(2)(3-bromoquinoline)(quinoline)] was prepared to alleviate the symmetry-imposed absence of inter-ring H2/H2 and H8/H8 NOESY cross-peaks for cis-[PtCl(2)(quinoline)(2)]. Molecular mechanics calculations on the complexes show the HTT rotamers to be 1-2 kJ mol(-)(1) more stable than the HTH forms, consistent with the (1)H spectra where the intensities of resonances for the two forms are approximately equal. Variable-temperature (1)H NMR spectra of cis-[PtCl(2)(quinoline)(2)] in dmf-d(7) indicate a rotational energy barrier of 82 +/- 4 kJ mol(-)(1). Variable-temperature (1)H NMR spectra indicate that the Br substituent on the quinoline ring does not affect the energy barrier to interconversion between the HTT and HTH forms (79 +/- 5 kJ mol(-)(1)). The steric contribution to the rotation barrier was calculated using molecular mechanics calculations and was found to be approximately 40 kJ mol(-)(1), pointing to a possible need for an electronic component to be included in future models.

Effects of trematode parasitism on the behaviour and ecology of a common marine snail (Littorina littorea (L.)).

Cryptocotyle lingua (Creplin) is a digenean trematode parasite of the littoral prosobranch gastropod Littorina littorea (L.). The literature suggests the snails become infected by grazing guano of the final host, the herring gull, Larus argentatus Pontoppidan. The parasite emerges from the snail as free-swimming cercariae. Interactions between the snail and the parasite at cellular and life-history levels are well established, but little is known of the influences the interaction has on the behaviour and the ecology of the snail. We tested the response of the snail to encounters with cercariae, examined the longevity of the guano on-shore and tested the responses of the snail to encounters with guano. Over half the L. littorea tested were able to detect both cercariae and a filtered homogenate of cercariae in conspecific mucus trails, approximately one-third of animals refusing to cross the treatments. Chemoreception by the mouth or foot is considered the most likely means of detection. Guano samples (mean weight 3.22 g) naturally deposited at approximately mid-tide level were completely washed away by one tidal inundation. We consider this period too brief to allow for ingestion of eggs in guano by the snail. Further, snails would not cross guano placed in conspecific trails. Most snails would not cross guano diluted by 10(3)x(10 mg ml(-1)) and some snails could still detect guano diluted by 10(6)x(10 &mgr;g ml(-1)), though all were prepared to cross it. Detection of guano is again believed to be by chemoreception by the mouth or foot. These results are discussed in terms of the mating and aggregating behaviour of L. littorea. Ingestion of the parasite by L. littorea is likely to take place once the guano has washed away as the eggs are negatively buoyant in seawater and may adhere to rock (biofilm) or algal fronds which may be grazed by the snail.

Rates of platination of -AG- and -GA- containing double-stranded oligonucleotides: effect of chloride concentration.

The kinetics of the reactions between 15N-labelled cisplatin and 14-base pair duplex oligonucleotides with either 5'-AG-3' or 5'-GA-3' groupings as the principal platination site are examined in the presence of 60-80 mM chloride by [1H,15N]HSQC 2D NMR spectroscopy. The presence of chloride at these concentrations results in a five-fold decrease in the rate of hydrolysis of cisplatin to cis-[PtCl(NH3)2(OH2)]+ and a two- to twenty-fold decrease in the rate of monofunctional adduct formation. The effects on the rate of closure from monofunctional to bifunctional adducts are less well established but some of these rates appear not to be significantly reduced by the presence of added chloride. The results provide a caution that the use of chloride to quench platination reactions may not be fully effective.

The orthopaedic management of peripheral ischaemia in meningococcal septicaemia in children.

Between March 1993 and February 1999, 14 children aged from eight months to 14.75 years were admitted to the paediatric intensive-care unit with meningococcal septicaemia in association with severe peripheral ischaemia. Of these, 13 were operated upon, eight of whom had early fasciotomies. Five children died. Of the nine survivors, one had no amputations while in the other eight 14 limb segments were amputated. We review the case histories and propose a protocol for the early management of these children.

Slowing of cisplatin aquation in the presence of DNA but not in the presence of phosphate: improved understanding of sequence selectivity and the roles of monoaquated and diaquated species in the binding of cisplatin to DNA.

1H-15N HSQC NMR spectroscopy is used to study the aquation reactions of cisplatin in 9 mM NaClO4 and 9 mM phosphate (pH 6) solutions at 298 K. For the first time in a single reaction and, therefore, under a single set of reaction conditions, the amounts of all species formed are followed and the rates of aquation, diaquation, and related anation processes are determined in both media. Binding of phosphate to aquated Pt species is observed, but the initial rate of aquation is not affected by the presence of 9 mM phosphate. The reaction between cisplatin and the 14-base-pair self-complementary oligonucleotide 5'-d(AATTGGTACCAATT)-3', having a GpG intrastrand binding site, is investigated. Various kinetic models for this reaction are evaluated and the most appropriate found to be that with a reversible aquation step and a single binding site for the self-complementary duplex. The rate constant for aquation is (1.62 +/- 0.02) x 10(-5) s-1, with the anation rate constant fixed at 4.6 x 10(-3) M-1 s-1, the value obtained from the aquation studies. The rate constants for monofunctional binding of cis-[PtCl(15NH3)2-(OH2)]+ to the sequence were 0.48 +/- 0.19 and 0.16 +/- 0.06 M-1 s-1 for the 3'- and 5'-guanine bases, respectively. Closure rate constants for the monofunctional adducts are (2.55 +/- 0.07) x 10(-5) and (0.171 +/- 0.011) x 10(-5) s-1, for the 3'- and 5'-guanines, respectively. The presence of DNA slows the aquation of cisplatin by 30-40% compared to that observed in 9 mM NaClO4 or 9 mM phosphate, and there is some evidence that the degree of slowing is sequence dependent. The possibility that cis-[Pt(OH)(NH3)2(OH2)]+ contributes to the binding of cisplatin to DNA is investigated, and it is found that about 1% followed this route, the majority of the binding occurring via the monoaquated species cis-[PtCl(NH3)2(OH2)]+. Comparison of the rates of disappearance of cisplatin in reactions at single defined GpG, ApG, GpA, GpTpG and 1,2-interstrand GG binding sites shows that the adduct profile is determined at the level of monofunctional adduct formation.

Arthroscopy of the hallux metatarsophalangeal joint.

Despite the paucity of literature on the subject of first MTP joint arthroscopy, the literature that does exist suggests that it is a worthwhile procedure in certain well-selected patients. These patients are usually young to middle-aged and have persistent pain and swelling of the first MTP joint that has failed to respond to conservative measures. These patients in general are too good for arthrodesis or arthroplasty, and certainly in the authors' experience, the results have been highly satisfactory.

Equilibrium and kinetic studies of the aquation of the dinuclear platinum complex [[trans-PtCl(NH3)2]2(mu-NH2(CH2)6NH2)]2+: pKa determinations of aqua ligands via [1H,15N] NMR spectroscopy.

By the use of [1H,15N] heteronuclear single quantum coherence (HSQC) 2D NMR spectroscopy and electrochemical methods we have determined the hydrolysis profile of the bifunctional dinuclear platinum complex [[trans-PtCl(15NH3)2]2(mu-15NH2(CH2)(6)15NH2)]2+ (1,1/t,t (n = 6), 15N-1), the prototype of a novel class of potential antitumor complexes. Reported are estimates for the rate and equilibrium constants for the first and second aquation steps, together with the acid dissociation constant (pKa1 approximately pKa2 approximately pKa3). The equilibrium constants determined by NMR at 25 and 37 degrees C (I = 0.1 M) were similar, pK1 approximately pK2 = 3.9 +/- 0.2, and from a chloride release experiment at 37 degrees C the values were found to be pK1 = 4.11 +/- 0.05 and pK2 = 4.2 +/- 0.5. The forward and reverse rate constants for aquation determined from this chloride release experiment were k1 = (8.5 +/- 0.3) x 10(-5) s-1 and k-1 = 0.91 +/- 0.06 M-1 s-1, where the model assumed that all the liberated chloride came from 1. When the second aquation step was also taken into account, the rate constants were k1 = (7.9 +/- 0.2) x 10(-5) s-1, k-1 = 1.18 +/- 0.06 M-1 s-1, k2 = (10.6 +/- 3.0) x 10(-4) s-1, k-2 = 1.5 +/- 0.6 M-1 s-1. The rate constants compare favorably with other complexes with the [PtCl(am(m)ine)3]+ moiety and indicate that the equilibrium of all these species favors the chloro form. A pKa value of 5.62 was determined for the diaquated species [[trans-Pt(15NH3)2(H2O)]2(mu-15NH2(CH2)(6)15NH2)]4+ (3) using [1H,15N] HSQC NMR spectroscopy. The speciation profile of 1 and its hydrolysis products under physiological conditions is explored.

Plantar fasciitis: how successful is surgical intervention?

Forty-three patients (47 heels) underwent decompression of the nerve to abductor digiti minimi with partial plantar fascia release for intractable plantar fasciitis over a 4-year period. Forty-one patients (45 heels) were available for follow-up. All of the patients had failed to respond to nonoperative treatment. The mean duration of symptoms before surgery was 34.8 months (range, 12-132 months), and the mean follow-up was 31.4 months (range, 11-66 months). Seventy percent of the patients in the study were overweight or obese. Before surgery, 39 patients (43 heels) rated their heel pain as severe. At follow-up, 34 of 45 (75.6%) of the heels were pain-free or only mildly painful. The mean visual analogue pain score dropped from 8.5 of 10 preoperatively to 2.5 of 10 postoperatively. Only four patients failed to report an improvement in their activity restrictions, and only one patient had a walking distance of under 100 m after surgery; this patient had been affected by a reflex sympathetic dystrophy. Overall, however, only 20 of 41 patients were totally satisfied with the outcome (48.8%). We recommend that the small group of patients who fail to respond to nonoperative treatment be considered for surgical intervention. The results in terms of symptomatic relief are generally good but in terms of patient satisfaction can only be rated as moderate. The patients should be counseled about the likely outcome of surgery.

Metatarsal neck osteotomy with rigid internal fixation for the treatment of lesser toe metatarsophalangeal joint pathology.

Metatarsalgia associated with metatarsophalangeal (MTP) joint instability and/or plantar callosity formation is a difficult problem to treat. During a 15-month period, we performed 50 osteotomies of the metatarsal neck with rigid internal fixation in 47 feet of 42 patients. Three patients were excluded from the study, leaving 47 osteotomies in 44 feet of 39 patients for review. There were 6 men and 33 women, with a mean age of 57 years. In addition to lesser MTP joint pain with or without instability, the majority of patients had first ray pathologic condition, which was also addressed at the time of surgery. All but one of the osteotomies were united radiologically at 6 weeks. The mean shortening was 4.1 mm (range, 2-12 mm), and the mean follow-up was 9 months. There were no cases of malunion, nonunion, or avascular necrosis. At follow-up, 33 patients were asymptomatic. Eight patients (nine feet) had a degree of persisting pain at follow-up (seven mild and two moderate), but the source of this pain was only the metatarsal or MTP joint that was operated on in three cases. In this article, we describe the indications, the technique, and the results of the osteotomy.

Intercuneiform instability and the "gap" sign.

We describe three cases of low-energy injuries to the midfoot resulting in rupture of Lisfranc's ligament without tarsometatarsal injury. Examination of the feet revealed an obvious physical sign only on weightbearing, and all three patients were noted at surgery to have intercuneiform instability in association with a rupture of Lisfranc's ligament.

Arthroscopy of the first metatarsophalangeal joint.

We carried out 12 arthroscopies of the first metatarsophalangeal (MTP) joint in 11 patients over a five-year period. Their mean age was 30 years (15 to 58) and the mean duration of symptoms before surgery was eight months (1 to 24). Six patients had an injury to the joint; all had swelling and tenderness with a reduced range of movement. In six patients, radiographs revealed no abnormality. Under general anaesthesia with a tourniquet the hallux is suspended by a large Chinese finger trap to distract the joint. Using a 1.9 mm 30 degree oblique arthroscope the MTP joint is inspected through dorsomedial and dorsolateral portals with a medial portal if necessary. All patients were found to have intra-articular pathology, which was treated using small instruments. The mean follow-up was 19.3 months (6 to 62) and all patients had no or minimal pain, decreased swelling and an increased range of movement of the affected joint.

Diatom motility: the search for independent replication of biological effects of extremely low-frequency electromagnetic fields.

PURPOSE: To investigate the hypothesis that exposure to a certain combination of static and time-varying electromagnetic fields (EMF) results in an increase in motility of the marine diatom Amphora coffeaeformis. MATERIALS AND METHODS: Diatom motility on agar was positively correlated with calcium ion (Ca2+) concentration. In previous experiments extremely low frequency EMF (16 Hz) had the greatest effect on diatoms at suboptimal for movement Ca2+ concentrations. To ensure that the required suboptimal Ca2+ concentration and resultant cell motility were found, a number of low concentrations of Ca2+ were experimentally tested. The EMF exposure conditions were those at the calculated calcium resonances (B(V)=0 microT; B(H)=20.9 microT; B(AC)=41.8 microT peak-peak 16 Hz) previously found to cause enhanced motility. Diatom movement on agar plates under EMF and control exposures was recorded. RESULTS: There was no significant difference in cell motility between control and EMF-exposed diatoms at each Ca2+ concentration tested. CONCLUSIONS: The study indicates that low-frequency EMF set at resonance conditions did not cause an increase in motility of the diatoms.

Diatom motility and low frequency electromagnetic fields--a new technique in the search for independent replication of results.

The hypothesis that exposure to a certain combination of static and alternating electromagnetic fields (EMFs) results in an increase in motility of the marine diatom Amphora coffeaeformis was tested. Diatom motility in three strains of A. coffeaeformis was positively correlated with extracellular calcium ion (Ca2+) concentration. The test apparatus consisted of two pairs of Helmholtz coils supported around the stage of a microscope linked to a video recorder and monitor. This system allowed real-time in vivo recordings of diatom speed under EMF and control exposures. The EMFs were calculated at calcium resonance values, previously found to cause enhanced motility. Computerised image analysis was used to calculate the distance moved by individual diatoms in 2-min periods before, during and after EMF or sham-EMF (control) exposure. The addition of EMF caused no significant increase in diatom motility. The results are discussed in relation to the use of diatom motility to measure EMF exposure effects.

Migration of the uncemented CLS acetabular component.

The migration of uncemented press-fit CLS (Cementless Total Hip Replacement System) acetabular cups was measured retrospectively from standard anteroposterior pelvic radiographs using a digitizer and software specifically developed for this study. A radiographic and clinical review also were undertaken. There were 96 patients in the study group. The mean follow-up for radiographic review was 2.23 years, and the mean follow-up for clinical review was 3 years. Migration was measured in the cranial and medial directions. Mean migration of the cup at 2 years was 1.35 mm in the cranial direction and 2.15 mm in the medial direction. Radiographic appearances altered little over the study period. Fifty-five (57.3%) patients demonstrated bone ingrowth onto the prosthesis, predominantly in DeLee and Charnley zone 1. Twenty-one patients demonstrated nonprogressive sclerotic lines; no lucent lines were encountered. Statistical analysis showed a significant correlation between acetabular ingrowth and reduced cup migration in the medial direction (P = .011 at 5 years). There was no statistical relationship between migration of the cup and radiographic features or clinical outcome.

Migration of the uncemented CLS femoral component.

The migration of uncemented CLS (Cementless Total Hip Replacement System) femoral stems was measured retrospectively from standard anteroposterior pelvic radiographs using a digitizer and software specifically designed for this study. The study population was comprised of 117 patients with 126 uncemented CLS femoral stems. All radiographs were reviewed and the incidence of thigh pain obtained by means of a questionnaire. The mean clinical follow-up was 3 years and the mean follow-up for radiographic review was 2.77 years. The mean femoral stem migration was 2 mm at 2 years and 3.66 mm at 7 years. Radiographic features varied little with no specific predictive features in patients with excessive early migration or those who complained of thigh pain. There was no statistical relationship between the presence of thigh pain and femoral stem migration.