Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

Kinetics of the Daily Rate of Photosynthesis at Low Temperatures for two Conifers

The daily course of photosynthesis at low temperatures in 2 coniferous species, Pinus ponderosa Laws., and Pseudotsuga menziesii (Mirb.) Franco, were studied using controlled environment facilities. After having been grown at a 23° day, and 19° night for a year, seedlings were acclimatized for 4 months to either a 3°, 7° or 11° day all under 1200 ft-c of light and followed by a 16-hour night at 3°. Measurement of photosynthesis at 1200 ft-c revealed 3 separate responses. First, the rapidity at which the plants attained their maximum photosynthesis when the lights were turned on depended upon the species, the current temperature, and the previous temperature condition to which the plants had become acclimatized. The warmer the day temperature the sooner the daily maximum was reached. Second, fluctuations in the rate of photosynthesis during the day varied with the species and the day temperature. Photosynthesis in both fir and pine kept at an 11° day and pines kept at a 7° day attained a daily peak rate followed by a decline. This decline occurred even though temperature and light were kept constant, the CO₂ level was returned to 320 ppm from 290 ppm, and the plants were kept well watered. At a 3° day neither species showed this decline. Third, a plant transferred to another temperature acquired a new stable daily photosynthetic pattern. The number of days required for stabilization depended upon the previous temperature history of the plant. The adjustment rate was faster when the temperature was raised than when it was lowered.     

Alpha Sarcin, a New Antitumor Agent: II. Fermentation and Antitumor Spectrum

Aspergillus giganteus MDH 18894 was found to produce alpha sarcin, a new antitumor agent inhibitory to a number of different animal tumors. This culture produced culture filtrates that inhibited mouse sarcoma 180 at dilutions up to 1:32. Optimal fermentation conditions were established for shake flasks and 30-liter and 100-gal (378.5-liter) fermentors. Results from a variety of media were reported; however, the medium which yielded the most reproducible filtrates consisted of 2% corn starch, 1.5% beef extract (Difco), 2% peptone (Difco), and 0.5% sodium chloride. Purified preparations of 800 sarcoma 180 dilution units per mg were inactive against bacteria, yeasts, molds, actinomycetes, and protozoa, but were cytotoxic to several strains of mammalian cells. Alpha sarcin inhibited sarcoma 180 in mice at a dose of 62.5 μg per kg per day. A variety of other animal tumors were also inhibited by this compound.     

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos^1,2 that we have successfully adapted^3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick⁵. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described⁶. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.To date, in utero electroporation has been most widely used for the analysis of neocortical development^1,2,3,4. More recent studies have targeted the embryonic retina^7,8,9 and thalamus^10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3^rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.Download video file.^{(82M, mov)}     

Clinical Manifestation of Depression after Stroke: Is It Different from Depression in Other Patient Populations?

Background

Despite ample research on depression after stroke, the debate continues regarding whether symptoms such as sleep disturbances, loss of energy, changes in appetite and diminished concentration should be considered to be consequences of stroke or general symptoms of depression. By comparing symptoms in depressed and non-depressed stroke patients with patients in general practice and patients with symptomatic atherosclerotic diseases, we aim to further clarify similarities and distinctions of depression after stroke and depression in other patient populations. Based on this, it is possible to determine if somatic symptoms should be evaluated in stroke patients in diagnosing depression after stroke.

Methods

An observational multicenter study is conducted in three hospitals and seven general practices including 382 stroke patients admitted to hospital with a clinical diagnosis of intracerebral hemorrhage or ischemic infarction, 1160 patients in general practice (PREDICT-NL), and 530 patients with symptomatic atherosclerotic diseases (SMART-Medea).

Results

The prevalence of major depressive disorder according to DSM-IV criteria was 14.1% (95% CI 11.0%-18.0%) in the stroke cohort, 5.4% (95% CI 3.8%-7.9%) in the symptomatic atherosclerotic diseases cohort and 12.9% (95% CI 11.1%-15.0%) in the general practice cohorts. Comparing depressed patients of the three cohorts demonstrated broadly similar symptom profiles, as well as comparable levels of individual symptom prevalence. However, the stroke patients suffered more severely from these symptoms than patients in the other populations.

Conclusions

The findings suggest that depression after stroke is not a different type of depression. This finding indicates that all depressive symptoms should be evaluated in stroke patients, including somatic symptoms.     

Detection of Heart Sounds in Children with and without Pulmonary Arterial Hypertension―Daubechies Wavelets Approach

Background

Automatic detection of the 1^st (S1) and 2^nd (S2) heart sounds is difficult, and existing algorithms are imprecise. We sought to develop a wavelet-based algorithm for the detection of S1 and S2 in children with and without pulmonary arterial hypertension (PAH).

Method

Heart sounds were recorded at the second left intercostal space and the cardiac apex with a digital stethoscope simultaneously with pulmonary arterial pressure (PAP). We developed a Daubechies wavelet algorithm for the automatic detection of S1 and S2 using the wavelet coefficient ‘D ₆’ based on power spectral analysis. We compared our algorithm with four other Daubechies wavelet-based algorithms published by Liang, Kumar, Wang, and Zhong. We annotated S1 and S2 from an audiovisual examination of the phonocardiographic tracing by two trained cardiologists and the observation that in all subjects systole was shorter than diastole.

Results

We studied 22 subjects (9 males and 13 females, median age 6 years, range 0.25–19). Eleven subjects had a mean PAP < 25 mmHg. Eleven subjects had PAH with a mean PAP ≥ 25 mmHg. All subjects had a pulmonary artery wedge pressure ≤ 15 mmHg. The sensitivity (SE) and positive predictivity (+P) of our algorithm were 70% and 68%, respectively. In comparison, the SE and +P of Liang were 59% and 42%, Kumar 19% and 12%, Wang 50% and 45%, and Zhong 43% and 53%, respectively. Our algorithm demonstrated robustness and outperformed the other methods up to a signal-to-noise ratio (SNR) of 10 dB. For all algorithms, detection errors arose from low-amplitude peaks, fast heart rates, low signal-to-noise ratio, and fixed thresholds.

Conclusion

Our algorithm for the detection of S1 and S2 improves the performance of existing Daubechies-based algorithms and justifies the use of the wavelet coefficient ‘D ₆’ through power spectral analysis. Also, the robustness despite ambient noise may improve real world clinical performance.     

Implementatie van een proactief zorgprogramma voor kwetsbare ouderen in de huisartsenpraktijk: van onderzoek naar praktijk

Background

Multimorbidity, functional impairment and frailty among community-dwelling older people are causing increasing complexity in primary care. A proactive integrated primary care approach is therefore essential. Between October 2014–2015, an evidence-based proactive care program for frail older people was implemented in the region Noord-West Veluwe en Zeewolde, the Netherlands. This study evaluated the feasibility of the implementation, having a strong focus on the collaboration between the medical and social domain.

Methods

Using a mixed-methods design we evaluated several process indicators. Data were obtained from electronic routine medical record data within primary care, questionnaires, and interviews with older adults. The questionnaires provided information regarding the expectations and experiences towards the program and were sent to health care professionals at baseline and six months follow-up. Stakeholders from various domains were asked to fill in the questionnaire at baseline and twelve months follow-up. Interviews were conducted to explore the experiences of older adults with the program. Regional work groups were set up in each municipality to enhance the interdisciplinary and domain transcending collaboration.

Results

The proactive primary care program was implemented in 42 general practices who provided care to 7904 older adults aged 75 years or older. A total of 101 health care professionals and 44 stakeholders filled in the questionnaires. The need for better structure and interdisciplinary cooperation seemed widespread among the participants. The implementation resulted in a positive significant change in the demand for a better regional healthcare-framework (34% p ≤ .001) among health care professionals, and the needs for transparency regarding the possibilities for referral improved (27% , p = .009). Half of the participants reported that the regional collaboration has been improved after the implementation. Health care professionals and stakeholders gained increased attention and awareness of frail elderly in their area compared to before the implementation. Older people and their caregivers were positive about the proactive approach. The nurses reported that the screenings questionnaire was too lengthy and therefore time consuming.

Conclusions

The implementation of the proactive primary care approach in daily practice was feasible. A strong interdisciplinary collaboration was realized. The program was easily adapted to the local context.

     

Prevention of depression and anxiety in adolescents: A randomized controlled trial testing the efficacy and mechanisms of Internet-based self-help problem-solving therapy

Background

Fractures of the long bones and femur fractures in particular are common in multiple trauma patients, but the optimal management of femur fractures in these patients is not yet resolved. Although there is a trend towards the concept of "Damage Control Orthopedics" (DCO) in the management of multiple trauma patients with long bone fractures as reflected by a significant increase in primary external fixation of femur fractures, current literature is insufficient. Thus, in the era of "evidence-based medicine", there is the need for a more specific, clarifying trial.

Methods/Design

The trial is designed as a randomized controlled open-label multicenter study. Multiple trauma patients with femur shaft fractures and a calculated probability of death between 20 and 60% will be randomized to either temporary fracture fixation with fixateur externe and defined secondary definitive treatment (DCO) or primary reamed nailing (early total care). The primary objective is to reduce the extent of organ failure as measured by the maximum sepsis-related organ failure assessment (SOFA) score.

Discussion

The Damage Control Study is the first to evaluate the risk adapted damage control orthopedic surgery concept of femur shaft fractures in multiple trauma patients in a randomized controlled design. The trial investigates the differences in clinical outcome of two currently accepted different ways of treating multiple trauma patients with femoral shaft fractures. This study will help to answer the question whether the "early total care" or the ?damage control” concept is associated with better outcome.

Trial registration

Current Controlled Trials ISRCTN10321620     

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.