Cloning and Expression Analysis of Human Amelogenin in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> Plants by Means of a Transient Expression System

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF–MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.     

Medical and psychological parameters in overweight and obese persons seeking treatment

The aim of the study was to analyse psychological characteristics and medical parameters in obese and overweight to identify the possible psychosocial consequences of obesity that may occur along with the numerous medical problems associated with excess body weight. Analysis was made on 296 patients (103 males and 193 females, median age 50, range 16-81) divided in three groups, depending on their Body mass index (BMI). Group I included 41 patients with BMI ranging from 25 to 29.9, group II included 170 patients with BMI from 30 to 34.9, and group III 85 patients with BM > or =35. We compared medical (glucose, cholesterol, triglycerides, HDL-cholesterol, systolic and diastolic blood pressure, body fat percentage) and psychological parameters (anxiety, depression, pros and cons of losing weight, self efficacy and four stages of change) in the patients included in the study. Univariate analysis has shown statistically significant difference among obese and overweight patients in goal weight, systolic and diastolic blood pressure, body fat percentage, glucose and cholesterol serum level. People with higher BMI (>30) found more advantages (pros) over disadvantages (cons) of weight loss but the level of anxiety and depression did not differ significantly among those 3 groups of patients. The results have shown that overweight and obese people have serious medical problems. They also differ in some psychological characteristics which have to be taken into consideration. Therefore, approach to these patients should be multidisciplinary, including dietary care, physical activity, psychological and medical care.     

Empathy in group psychoanalytic psychotherapy: questionnaire development

The aim of this study is to develop a questionnaire that can observe empathy in group psychoanalytic psychotherapy and examine the structure of its factors. A questionnaire comprised of 160 items in five-point Likert-type scale was developed through analysis of communication and interaction related to empathizing during group sessions. The questionnaire was applied on 256 patients from 40 therapy groups in 9 cities in Croatia. All 20 group analysts are trained in the Institute for Group Analysis in Zagreb. The patients were selected based on group analysis criteria. After item discrimination and principal component analysis limited to five factors were assessed, 80 items were isolated, 20 of which made a control scale for socially desirable responses. Two parallel questionnaire forms were developed: Group-Analysis-Empathy 1 (GA-Em1) and Group-Analysis-Empathy 2 (GA-Em2). A new, reliable and valid questionnaire for empathy observation employable in group psychotherapy was designed. The following factors were isolated by means of factor analysis: 1. Emotional disclosure and sensibility; 2. Containing and metabolizing; 3. Immersion; 4. Resonance and responsiveness; 5. Insight. A new questionnaire on empathy in group-analytical psychotherapy can measure the capacity for emotional communication among group members and between the group and the group analyst - conductor.     

High-performance liquid chromatographic determination of sphinganine and sphingosine in serum and urine of subjects from an endemic nephropathy area in Croatia

Endemic nephropathy (EN) is a chronic renal disease present as an endemic in Brodska Posavina, Croatia. The aim of the study was to assess the possible role of fumonisins, i.e., mycotoxins produced by Fusarium moniliforme, as causative agents for EN. Fumonisins inhibit ceramide synthase, the enzyme of de novo synthesis of sphingolipids, which leads to an increase in the sphinganine/sphingosine ratio. In the present study, a modified method has been used for the determination of the sphinganine/sphingosine ratio in human serum and urine of healthy subjects and EN patients from the endemic area. Free sphingoid bases, sphinganine and sphingosine, were obtained by base hydrolysis. Afterwards, precolumn ortho-phthaldialdehyde derivatisation, HPLC separation and quantification by fluorescence detection were performed. The results thus obtained pointed to a sphingolipid metabolism impairment, which may have been induced by fumonisins or fumonisin-like mycotoxins. As statistically significant differences were recorded in the subjects not yet affected with EN, an impairment in the metabolism of sphingolipids might be considered as an early indicator of EN.     

A single aquaporin gene encodes a water/glycerol/urea facilitator in Toxoplasma gondii with similarity to plant tonoplast intrinsic proteins

We describe a single aquaporin gene in Toxoplasma gondii which, surprisingly, has only 28% sequence similarity to the aquaglyceroporin of another apicomplexan parasite, Plasmodium falciparum. Sequence comparisons showed 47% similarity to water-specific plant aquaporins and the conservation of typical pore-forming residues. We established that the Toxoplasma aquaporin protein is a bifunctional membrane pore with intermediate water and high glycerol permeability. Furthermore, we identified hydroxyurea, an antineoplastic agent with inhibitory effects on parasite proliferation, as a permeant of this channel.     

Patients' ranking of therapeutic factors in group analysis

The aim of this research is to assess which therapeutic factors are of greatest importance to patients in group analytic psychotherapy, and whether the patients' characteristics and the phase of the group process influenced their evaluation of therapeutic factors. The Yalom's group therapeutic factors questionnaire was filled out by 66 patients, members of small groups conducted according to group analytic principles. The average scores for each therapeutic factor were subsequently ranked by importance to the patients and related to their age, sex, education, previous psychotherapeutic experience and phase of group process. Self-understanding was the highest-ranking therapeutic factor for the patients (average score 21.32 +/- 0.04 out of 25 maximum), whereas identification was the lowest ranking factor (15.88 +/- 0.06 in average). Group therapeutic factors were scored higher by women, patients up to 30 years of age, high-school graduates, and those with previous psychotherapeutic experience. Self-understanding seems to be the most important therapeutic factor in group analysis, emphasizing the importance of appropriate selection of patients for group analysis in order to utilize therapeutic factors the best.