Isolation and characterization of human smooth muscle cells from umbilical cord vein and their reconstitution in a vascular co-culture model with underlying endothelial cells

Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against smooth muscle actin, negative for antibodies against factor VIII and displayed typical hill and valley morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×10⁴ cells/cm² for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 M), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.Abbreviations BCA bicinchoninic acid - BSA bovine serum albumin - DMEM DMEM medium supplemented with 4.5 g/l glucose, 100 IU/ml penicillin, 100 g/ml streptomycin and 1.25 g/ml fungizone - DMEM-1 DMEM supplemented with 20% FBS, 300 IU/ml penicillin, 300 g/ml streptomycin and 3.75 mg/ml fungizone - DMEM-2 DMEM supplemented with 20% FBS, 100 IU/ml of penicillin, 100 g/ml of streptomycin and 1.25 g/ml of fungizone - DMSO dimethyl sulfoxide - FBS fetal bovine serum - HPF human pulmonary fibroblasts - HS human serum - HUVE human umbilical vein endothelial - HUVSM human umbilical vein smooth muscle - M199 M199 medium supplemented with 20% HS, 100 IU/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml fungizone and 2 mM L-glutamine - P passage number - PBS phosphate buffered saline - TCA trichloroacetic acid - vwF von Willebrand factor (factor VIII)     

Cardiac Orienting to Pulsed and Continuous Auditory Stimulation: A Developmental Study

Pulsed presentations of auditory stimulation have been shown to be particularly effective in eliciting cardiac deceleration (orienting) during earliest infancy. In the present study the processing of pulsed as compared to continuous stimulation was studied developmentally for two infant age groups (13–18 and 26–35 weeks) and one adult group. Ten habituation trials of either a pulsed or a continuous 10-sec tone were followed by 2 dishabituation trials, in which the temporal pattern (continuous vs pulsed) was shifted. The dishabituation data showed that the two kinds of stimulation were discriminated, but the only finding of differential processing relating to the relevance of pulsed stimulation for younger infants was a tendency for infant subjects, unlike adults, to show prolonged orienting to initial presentations of the pulsed tone. For rate of habituation there were no differential effects. Only in the adult group was the response during dishabituation influenced by the direction of change of temporal pattern. Orienting to stimulus offset was observed on initial presentations of the continuous stimulus for the older infant group.     

Selective Attention Effects on the Reflex Blink

Past work has shown that facilitation of reflex blinking accompanies cardiac deceleration when the attention of subjects is directed to a reflex-eliciting stimulus. The present studies showed that when warning stimuli directed attention instead to weak (tactile) stimuli presented simultaneously with reflex-eliciting (acoustic) stimuli, cardiac deceleration was still present but reflex magnitude was unchanged or inhibited. However, latency to reflex onset remained facilitated, i.e., latency and magnitude changes were discordant. The findings were interpreted as evidence for two independent processes: a process capable of selectively enhancing or attenuating sensory input and a non-selective process presumably facilitating motor pathways.     

Interaction between mothers and infants born at risk during the first six months of corrected age

Abstract The effects of preterm birth and the perinatal infant health condition on mother-infant interactions were analysed in 278 mother-infant pairs, divided into four groups according to infants' gestational age at birth: group 1. 23–31 weeks; group 2,32–36 weeks; group 3, 37–42 weeks; and group 4, a control group of healthy full-term infants. The methodological approach was based on observation of the pairs at 2,4 and 6 months of infants" corrected age (± 1 week) during undressing of the infant and face-to-face interaction. It was found that mother-infant pairs with preterm infants (groups 1 and 2) did not differ in interactional variables from those of the control group. On the other hand, the birth of a full-term infant in need of neonatal intensive care (group 3) affected maternal and infant interactive behaviour. Additionally, infants from group 3 did not show stability in their interactive behaviour between any ages of measurement. This result suggests that interactive behaviour of full-term infants in need of neonatal intensive care are rather unpredictable during their first 6 months of life, which might have contributed to the less optimal interactive pattern observed for their mothers compared with mothers of the control group.     

Inhibition and executive functioning in Type A and ADHD boys

The present study was aimed at clarifying the standing of Type A behavior, as measured by behavioral observations, relative to Attention Deficit Hyperactivity Disorder (ADHD), using measures of inhibitory control and executive functioning. The study sample included 20 boys exhibiting Type A behavior, 21 boys exhibiting Type B behavior and 14 boys diagnosed with ADHD, ranging in age from 7 to 12 years. The results of the present study showed that the Type A children differed from Type B children on two time-related variables, response latency and reaction time, in accordance with the view of Type As as time-urgent and impatient. Furthermore, in comparisons with the ADHD group, the Type A boys were found to be superior on several performance tasks, such as Go/no-go omissions, time reproduction, story recall and memory for sequences of hand movements, although similarities between Type A and ADHD boys were evidenced in terms of response latency and reaction time. In other words, although Type A boys were similar to ADHD boys in terms of overt displays of time-urgency and impatience, Type As do not display deficits with regard to executive functioning, of the kind often found when ADHD children are compared with normal controls. It may thereby be concluded that Type A behavior and hyperactivity/ADHD appear to be well differentiated except with regard to what may be interpreted as impatience. Speculations concerning differing origins of overtly similar characteristics of Type A behavior and ADHD should be considered in future research.     

Clinical course and renal function in minimal change nephrotic syndrome

Thirteen unselected children with the minimal change nephrotic syndrome were followed for 11 months-7.5 years, during which time 32 renal function tests were performed. Glomerular filtration rate (GFR) was determined as clearance of inulin using a standard clearance technique. The children had between zero and 34 relapses and three developed steroid resistance. Three patients had severe but reversible complications related to the corticosteroid therapy. Chlorambucil was given to four patients, of whom three with steroid resistance had lasting remissions, whereas one patient with frequent relapses had only transient improvement. The GFR was decreased at the first episode in six patients, whose clinical course did not differ from that of the others. None had a decreased GFR later in the course of the disease. It is concluded that reduced GFR at the onset of the minimal change nephrotic syndrome is reversible and does not imply an unfavourable outcome.     

Functional versus chemical diversity: is biodiversity important for drug discovery?

Prospecting the full biodiversity of nature to find leads for new drugs is not necessary. Because finding leads is aimed at identifying biological activity, structure is of secondary importance. Furthermore, although natural chemical diversity might be unrivalled, functional diversity is bound to be considerably less. It is likely that many millions of chemically distinct molecules exist in nature but it is inconceivable that the number of different biological functions is near this number. This is corroborated by knowledge obtained from the genome sequences of an increasing number of species. It is unlikely that ligands for specific molecular targets are restricted to one species and even individual compounds are often found in more than one species. Important molecular mechanisms are likely to be ubiquitous and there are no a priori reasons to assume that some are restricted to, for example, tropical rainforests. Thus, there are no obvious advantages of "biodiversity prospecting", which will, possibly, endanger fragile ecosystems in the search for rare species.     

Reversible antifouling effect of the cyclotide cycloviolacin O2 against barnacles

Cycloviolacin O2, a plant peptide of the cyclotide family, is shown to have potent effects against fouling barnacles (Balanus improvisus), with complete inhibition of settlement at a concentration of 0.25 microM. The effect of cycloviolacin O2 against barnacles is reversible and nontoxic in the bioassay employed in these studies. Cycloviolacin O2 was isolated from the terrestrial plant Viola odorata by strong cation exchange and reversed-phase HPLC and identified by mass spectrometry following aminoethylation and enzymatic cleavage.