Recovery of Yeast Saccharomyces cerevisiae after Thermal Insult

All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.

Heat shock response in mulberry silkworm races with different thermotolerances

The thermal sensitivity and heat shock response of the different races of the mulberry silkworm Bombyx mori have been analysed. The multivoltine race, strains C. Nichi and Pure Mysore showed better survival rates than the bivoltine race, strain NB4D2 exposed to 41 degrees C and above. In general, the fifth instar larvae and the pupae exhibited maximum tolerance compared to the early larval instars, adult moths or the eggs. Exposure up to 39 degrees C for 1 or 2 h was tolerated equally whereas temperatures above 43 degrees C proved to be lethal for all. Treatment of larvae at 41 degrees C for Ih resulted in a variety of physiological alterations including increased heart beat rates, differential haemocyte counts, enlargement of granulocytes and the presence of additional protein species in the tissues and haemolymph. The appearance of a 93 kDa protein in the haemolymph, fat bodies and cuticle, following the heat shocking of larvae in vivo was a characteristic feature in all the three strains examined although the kinetics of their appearance itself was different. In haemolymph, the protein appeared immediately in response to heat shock in C. Nichi reaching the maximal levels in 2-4 h whereas its presence was noticeable only after 2-4 h recovery time in Pure Mysore and bivoltine races. The fat body from both C. Nichi and NB4D2 showed the presence of 93 kDa, 89 kDa and 70 kDa proteins on heat shock. The haemocytes, on the other hand, expressed only a 70 kDa protein consequent to heat shock. The 93 kDa protein in the haemolymph, therefore could have arisen from some other tissue, possibly the fat body. The 93 kDa protein was detected after heat shock in pupae and adult moths as well, although the presence of an additional (56 kDa) protein was also apparent in the adults. The presence of 46 kDa and 28 kDa bands in addition to the 93 kDa band in the cuticular proteins immediately following heat shock was clearly discernible. The 70 kDa band did not show much changes in the cuticular proteins on heat shock. In contrast to the changes in protein profiles seen in tissues and haemolymph following heat shock in vivo, the heat treatment of isolated fat body or haemolymph in vitro resulted in protein degradation.

In vivo treatment of Heymann's Nephritis using a cytotoxic protein-toxin conjugate

In this study we investigated the possibility of treating Heymann's Nephritis (HN) by destroying antibody producing cells by targetting a toxin, gelonin - conjugated to gp330, the renal brush border antigen. HN was induced in rats by immunizing them with purified gp330. The gelonin-gp330 conjugate was administered 12 days after the antigenic challenge. Serum was screened for circulating antibodies. Proteinurea was estimated. The gp330-gelonin conjugate-treated animals had a circulating antibody titre in the serum much lower than that of diseased (untreated) animals. Proteinurea seen in diseased animals was not observed in treated animals. This work suggests the possibility of using a toxin-antigen conjugate for immunomodulating antibody mediated autoimmune renal disease.

Corneal recovery after uncomplicated and complicated PRK and LASIK

Refractive errors, especially myopia, seem to increase worldwide. Concurrently, the number of surgical refractive corrections has increased rapidly, with several million procedures performed annually. However, excimer laser surgery was introduced after a limited number of studies done with animals and to date there still are only few long-term follow-up studies of the results. The present thesis aims to evaluate the safety and functional outcome of, as well as to quantify the cellular changes and remodelling in the human cornea after, photorefractive keratectomy (PRK) and laser assisted in situ keratomileusis (LASIK). These procedures are the two most common laser surgical refractive methods. In Study I, myopic ophthalmic residents at Helsinki University Eye Hospital underwent a refractive correction by PRK. Five patients were followed up for 6 months to assess their subjective experience in hospital work and their performance in car driving simulator and in other visuomotor functions. Corneal morphological changes were assessed by in vivo confocal microscopy (ivCM). Study II comprised 14 patients who had undergone a PRK operation in 1993-1994. Visual acuity was examined and ivCM examinations performed 5 years postoperatively. In Study III 15 patients received LASIK refractive correction for moderate to high myopia (-6 - -12 D). Their corneal recovery was followed by ivCM for 2 years. Diffuse lamellar keratitis (DLK) is a common but variable complication of LASIK. Yet, its aetiology remains unknown. In Study IV we examined six patients who had developed DLK as a consequence of formation of an intraoperative or post-LASIK epithelial defect, to assess the corneal and conjunctival inflammatory reaction. In the whole series, the mean refractive correction was -6.46 diopters. The best spectacle corrected visual acuity (BSCVA) improved in 30 % of patients, whereas in four patients BSCVA decreased slightly. The mean achieved refraction was 0.35 D undercorrected. After PRK, the stromal scar formation was highest at 2 to 3 months postoperatively and subsequently decreased. At 5 years increased reflectivity in the subepithelial stroma was observed in all patients. Interestingly, no Bowman s layer was detected in any patient. Subbasal nerve fiber bundle(snfb) regeneration could be observed already at 2 months in 2 patients after PRK. After 5 years, all corneas presented with snfb, the density of which, however, was still lower than in control corneas. LASIK induced a hypocellular area on both sides of the flap interface. A decrease of the most anterior keratocyte density was also observed. In the corneas that developed DLK, inflammatory cell-type objects were present in the flap interface in half of the patients. The other patients presented only with keratocyte activation and highly reflective extracellular matrix. These changes resolved completely with medication and time. Snfb regeneration was first detected at one month post-LASIK, but still after two years the density of snfb, however, was only 64 % of the preoperative values. The performance of ophthalmological examinations and microsurgery without spectacles was easier postoperatively, which was appreciated by the residents. Both PRK and LASIK showed moderate to good accuracy and high safety. In terms of visual perception and subjective evaluation, few patients stated any complaints in the whole series of studies. Instead, the majority of patients experienced a marked improvement in everyday life and work performance. PRK and LASIK have shown similar results, with good long term morphological healing. It seems evident that, even without the benefit of over-20-year follow-up results, these procedures are sufficiently safe and accurate for refractive corrections and corneal reshaping.

Microsurgical Aneurysm Model in Rats and Mice: Development of Endovascular Treatment and Optimization of Magnetic Resonance Imaging

The rupture of a cerebral artery aneurysm causes a devastating subarachnoid hemorrhage (SAH), with a mortality of almost 50% during the first month. Each year, 8-11/100 000 people suffer from aneurysmal SAH in Western countries, but the number is twice as high in Finland and Japan. The disease is most common among those of working age, the mean age at rupture being 50-55 years. Unruptured cerebral aneurysms are found in 2-6% of the population, but knowledge about the true risk of rupture is limited. The vast majority of aneurysms should be considered rupture-prone, and treatment for these patients is warranted. Both unruptured and ruptured aneurysms can be treated by either microsurgical clipping or endovascular embolization. In a standard microsurgical procedure, the neck of the aneurysm is closed by a metal clip, sealing off the aneurysm from the circulation. Endovascular embolization is performed by packing the aneurysm from the inside of the vessel lumen with detachable platinum coils. Coiling is associated with slightly lower morbidity and mortality than microsurgery, but the long-term results of microsurgically treated aneurysms are better. Endovascular treatment methods are constantly being developed further in order to achieve better long-term results. New coils and novel embolic agents need to be tested in a variety of animal models before they can be used in humans. In this study, we developed an experimental rat aneurysm model and showed its suitability for testing endovascular devices. We optimized noninvasive MRI sequences at 4.7 Tesla for follow-up of coiled experimental aneurysms and for volumetric measurement of aneurysm neck remnants. We used this model to compare platinum coils with polyglycolic-polylactic acid (PGLA) -coated coils, and showed the benefits of the latter in this model. The experimental aneurysm model and the imaging methods also gave insight into the mechanisms involved in aneurysm formation, and the model can be used in the development of novel imaging techniques. This model is affordable, easily reproducible, reliable, and suitable for MRI follow-up. It is also suitable for endovascular treatment, and it evades spontaneous occlusion.

Ibuprofen ingestion does not affect markers of post-exercise muscle inflammation

Purpose: We investigated if oral ingestion of ibuprofen influenced leucocyte recruitment and infiltration following an acute bout of traditional resistance exercise Methods: Sixteen male subjects were divided into two groups that received the maximum over-the-counter dose of ibuprofen (1200mg d−1) or a similarly administered placebo following lower body resistance exercise. Muscle biopsies were taken from m.vastus lateralis and blood serum samples were obtained before and immediately after exercise, and at 3 and 24 h after exercise. Muscle cross-sections were stained with antibodies against neutrophils (CD66b and MPO) and macrophages (CD68). Muscle damage was assessed via creatine kinase and myoglobin in blood serum samples, and muscle soreness was rated on a ten-point pain scale. Results: The resistance exercise protocol stimulated a significant increase in the number of CD66b+ and MPO+ cells when measured 3 h post exercise. Serum creatine kinase, myoglobin and subjective muscle soreness all increased post-exercise. Muscle leucocyte infiltration, creatine kinase, myoglobin and subjective muscle soreness were unaffected by ibuprofen treatment when compared to placebo. There was also no association between increases in inflammatory leucocytes and any other marker of cellular muscle damage. Conclusion: Ibuprofen administration had no effect on the accumulation of neutrophils, markers of muscle damage or muscle soreness during the first 24 h of post-exercise muscle recovery.

Immediate Breast Reconstructions in the Treatment of Breast Cancer

Breast cancer is the most common form of potentially fatal cancer in women in the Western world. Better understanding of the breast cancer disease process together with developments in treatments have led to improved survival and reduced risk of recurrence, significantly influencing the acceptance of breast reconstructions as part of breast cancer treatment. Skin-sparing mastectomy followed by immediate breast reconstruction has proved superior to other forms of breast reconstruction in terms of aesthetic outcome. However, due to the relatively recent introduction of skin-sparing mastectomy concerns on the surgical and oncological safety of the operation persist. The aim of the present study is to evaluate the surgical and oncological safety of skin-sparing mastectomy and immediate breast reconstruction in a consecutive patient series with ensuing follow-up. Subsequent aims of the study are to examine possibilities of reducing surgical complications of the operation and to assess the feasibility of sentinel node biopsy together with immediate breast reconstruction. The study population comprises a consecutive series of patients having undergone skin-sparing mastectomy followed by immediate breast reconstruction at the Helsinki University Central Hospital between 1992 and 2006. In Study I, the hospital records of 207 patients, operated between 1992 and 2001, were analyzed for surgical complications and recurrences of breast cancer during follow-up. In Study II, 60 consecutive patients were randomized into either conventional diathermy or radiofrequency coagulation groups to examine possibilities of reducing skin-flap complications. Study III consists of 62 consecutive breast cancer patients evaluated for the feasibility of sentinel node biopsy simultaneously with immediate breast reconstruction. In Study IV, hospital records were analyzed to examine local recurrence of breast cancer in a consecutive series of 146 patients with Stage I or II disease. Post-operative complications in Study I included native skin-flap necrosis (10.1%), hematoma (10.1%), anastomose thrombosis (5.3%), infection (3.4%), hernia (2.6%) and loss of one microvascular flap (0.7%). The Stage I and II patients in Study IV had a local recurrence rate of 2.7%, an isolated regional lymph node recurrence rate of 2.1% and a systemic recurrence rate of 2.7%, during a mean follow-up time of 51 months. The Stage III patients in study I had a locoregional recurrence rate of 31.3% during follow-up. Radiofrequency coagulation in Study II did not decrease skin-flap complications when compared with conventional diathermy. An increased skin-flap complication rate in Study II was associated with smoking and the type of skin incision used. In Study III, eleven patients had tumor positive sentinel nodes, nine of which were detected intraoperatively. Skin-sparing mastectomy followed by immediate breast reconstruction is a safe procedure both surgically and oncologically, especially for early stage breast cancer. Tennis racket type incision is associated with an increased skin-flap complication rate. Sentinel node biopsy with intraoperative assessment of sentinel node metastases is feasible in patients undergoing immediate breast reconstruction.

Epidemiology and Treatment Options of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is caused by an autoimmune inflammation of the small bile ducts. It results to destruction of bile ducts, accumulation of the bile in the liver, and cirrhosis. The prevalence and incidence of PBC is increasing in the Western world. The prevalence is highest in the USA (402 per million) and incidence in Scotland (49/million/year). Our aim was to assess the epidemiology of PBC in Finland. Patients for the epidemiological study were searched from the hospital discharge records from year 1988 to 1999.The prevalence rose from 103 to 180/million from 1988 to 1999, an annual increase of 5.1%. The incidence rose from 12 to 17 /million/year, an annual increase of 3.5%. The age at death increased markedly from 65 to 76 years. The risk of liver related deaths diminished over time. The treatment of PBC is based on Ursodeoxycholic acid (UDCA). During 20 years 50% of patients end up with cirrhosis. Our treatment option was to combine budesonide, a potent corticosteroid with a high first pass metabolism in the liver, to UDCA and evaluate the liver effects and systemic effects such as bone mass density (BMD) changes. Our aim was to find out if combination of laboratory tests would serve as a surrogate marker for PBC and help reducing the need for liver biopsy. Non-cirrhotic PBC patients were randomized to receive budesonide 6 mg/day combined to UDCA 15 mg /kg/day or UDCA alone for three years. The combination therapy with UDCA and budesonide was effective: stage improved 22%, fibrosis 25%, and inflammation 32%. In the UDCA group the changes were: 20% deterioriation in stage and 70% in fibrosis, but a 10% improvement in inflammation. BMD in femoral neck decreased by 3.6% in the combination group and by 1.9% in the UDCA group. The reductions in lumbar spine were 2.8% and 0.7%. Pharmacokinetics did not differ between the stages of PBC. HA, PIIINP, bile acids, and AST were significantly different within stages I-III and could differentiate the mild fibrosis (F0F1) from the moderate (F2F3). The combination of these individual markers (PBC-score) further improved the accuracy. The area under the ROC of the PBC score, using a cut of value 66, had a sensitivity of 81.4% and a specificity of 65.2% to classify the stage of PBC. The prevalence of PBC in Finland increases, which results from increasing incidence and improved survival. The combination of budesonide and UDCA improves liver histology compared to UDCA alone in non-cirrhotic stages of PBC. The treatment may reduce BMD. Hyaluronic acid, PIIINP, AST, and bile acids may serve as tools to monitor the treatment response in the early stages of PBC. The budesonide and UDCA combination therapy is an option for those patients who do not receive full response from UDCA and are still at the non-cirrhotic stage of PBC.

Outcome of singleton pregnancy after assisted reproductive treatment

Singleton pregnancies achieved by means of assisted reproductive treatment (ART) are associated with increased obstetric and neonatal risks in comparison with spontaneously conceived singleton pregnancies. The impact of infertility- and treatment-related factors on these risks is not properly understood. In addition, the psychological effects of infertility and its treatment on the experience of pregnancy have scarcely been studied. Thus, the aim of the present study was to evaluate the importance of infertility- and treatment-related factors on prediction of pregnancy outcome, obstetric and neonatal risks, fear-of-childbirth and pregnancy-related anxiety. The subjects consisted of infertile women who achieved a singleton pregnancy by means of in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). The control groups comprised spontaneously conceiving women with singleton gestations. Early pregnancy outcome was assessed by means of assay of serum human chorionic gonadoptrophin (hCG) in single samples. Other outcome data were collected from patient records, national Health Registers and via prospective questionnaire surveys. Viable pregnancies were associated with significantly higher serum hCG levels 12 days after embryo transfer than non-viable pregnancies. Among singleton pregnancies, aetiological subgroup, treatment type or the number of transferred embryos did not impair the predictive value of single hCG assessment. According to the register-based data, age-, parity- and socioeconomic status- adjusted risks of gestational hypertension, preterm contractions and placenta praevia were more frequent in the ART pregnancies than in the control pregnancies. Significantly higher rates of induction of delivery and Caesarean section occurred in the ART group than in the control group. The risks of preterm birth and low birth weight (LBW) were increased after ART pregnancy. Duration or aetiology of infertility, treatment type (fresh or frozen IVF or ICSI) or rank of treatment did not contribute to the risks of preterm birth or LBW. In addition, the risks of preterm birth and LBW remained elevated in spite of of the number of transferred embryos. Although mean duration of pregnancy was shorter and mean birth weight lower in the ART pregnancies than in the control pregnancies, these differences were hardly of clinical significance. Fear-of-childbirth and pregnancy-related anxiety were equally common to women conceiving by means of ART, or spontaneously. Partnership of five to ten years appeared to be protective as regards severe fear-of-childbirth, whereas long preceding infertility (≥ seven years) had the opposite effect. In conclusion, an early hCG assessment maintained its good predictive value regardless of infertility- or patient-related factors. Further, we did not recognise any infertility- or patient-related factors that would expose infertile women to increased obstetric or neonatal risks. However, a long period of infertility was associated with severe fear-of-childbirth.

Effects of unilateral vocal fold paralysis treatment with fascia augmentation on airflow mechanics, tracheal sounds and voice acoustics

Data on the influence of unilateral vocal fold paralysis on breathing, especially other than information obtained by spirometry, are relatively scarce. Even less is known about the effect of its treatment by vocal fold medialization. Consequently, there was a need to study the issue by combining multiple instruments capable of assessing airflow dynamics and voice. This need was emphasized by a recently developed medialization technique, autologous fascia injection; its effects on breathing have not previously been investigated. A cohort of ten patients with unilateral vocal fold paralysis was studied before and after autologous fascia injection by using flow-volume spirometry, body plethysmography and acoustic analysis of breathing and voice. Preoperative results were compared with those of ten healthy controls. A second cohort of 11 subjects with unilateral vocal fold paralysis was studied pre- and postoperatively by using flow-volume spirometry, impulse oscillometry, acoustic analysis of voice, voice handicap index and subjective assessment of dyspnoea. Preoperative peak inspiratory flow and specific airway conductance were significantly lower and airway resistance was significantly higher in the patients than in the healthy controls (78% vs. 107%, 73% vs. 116% and 182% vs. 125% of predicted; p = 0.004, p = 0.004 and p = 0.026, respectively). Patients had a higher root mean square of spectral power of tracheal sounds than controls, and three of them had wheezes as opposed to no wheezing in healthy subjects. Autologous fascia injection significantly improved acoustic parameters of the voice in both cohorts and voice handicap index in the latter cohort, indicating that this procedure successfully improved voice in unilateral vocal fold paralysis. Peak inspiratory flow decreased significantly as a consequence of this procedure (from 4.54 ± 1.68 l to 4.21 ± 1.26 l, p = 0.03, in pooled data of both cohorts), but no change occurred in the other variables of flow-volume spirometry, body-plethysmography and impulse oscillometry. Eight of the ten patients studied by acoustic analysis of breathing had wheezes after vocal fold medialization compared with only three patients before the procedure, and the numbers of wheezes per recorded inspirium and expirium increased significantly (from 0.02 to 0.42 and from 0.03 to 0.36; p = 0.028 and p = 0.043, respectively). In conclusion, unilateral vocal fold paralysis was observed to disturb forced breathing and also to cause some signs of disturbed tidal breathing. Findings of flow volume spirometry were consistent with variable extra-thoracic obstruction. Vocal fold medialization by autologous fascia injection improved the quality of the voice in patients with unilateral vocal fold paralysis, but also decreased peak inspiratory flow and induced wheezing during tidal breathing. However, these airflow changes did not appear to cause significant symptoms in patients.