CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.

Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting ß2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Age differences in central auditory system responses to naturalistic music.

Aging influences the central auditory system leading to difficulties in the decoding and understanding of overlapping sound signals, such as speech in noise or polyphonic music. Studies on central auditory system evoked responses (ERs) have found in older compared to young listeners increased amplitudes (less inhibition) of the P1 and N1 and decreased amplitudes of the P2, mismatch negativity (MMN), and P3a responses. While preceding research has focused on simplified auditory stimuli, we here tested whether the previously observed age-related differences could be replicated with sounds embedded in medium and highly naturalistic musical contexts. Older (age 55-77 years) and younger adults (age 21-31 years) listened to medium naturalistic (synthesized melody) and highly naturalistic (studio recording of a music piece) stimuli. For the medium naturalistic music, the age group differences on the P1, N1, P2, MMN, and P3a amplitudes were all replicated. The age group differences, however, appeared reduced with the highly compared to the medium naturalistic music. The finding of lower P2 amplitude in older than young was replicated for slow event rates (0.3-2.9 Hz) in the highly naturalistic music. Moreover, the ER latencies suggested a gradual slowing of the auditory processing time course for highly compared to medium naturalistic stimuli irrespective of age. These results support that age-related differences on ERs can partly be observed with naturalistic stimuli. This opens new avenues for including naturalistic stimuli in the investigation of age-related central auditory system disorders.

Influence of Von Willebrand Disease (VWD) and pregnancy on the expression of angiogenic factors in the porcine female reproductive tract.

Von Willebrand Disease (VWD) is a heritable disorder caused by defects of the Von Willebrand Factor (VWF), leading to deficiencies in coagulation and also angiogenesis. Women affected by VWD frequently show bleeding concerning the reproductive tract and may present with increased rates of miscarriages. We used a porcine model representing VWD type 1 and type 3 as well as the wildtype. Samples were obtained from the reproductive tract of non-pregnant sows and sows pregnant at time of placentation. Relative expression of the genes CALR, CCN2, CXCL8, ECE1, EDN1, F8, IGFBP7, and LGALS3 was analyzed. CCN2 and FVIII proteins were additionally analyzed using immunohistochemistry. In uterus and ovary significant upregulation of CCN2 was seen in non-pregnant pigs affected by VWD. This might be caused by the higher VEGFA-levels in these pigs and could have an influence angiogenesis. During pregnancy, CCN2 expression increased in wildtype pig uteri but hardly changed in those of pregnant pigs affected by VWD, presumably because the expression level in the latter pigs already was significantly increased before pregnancy. F8 expression was significantly reduced in uterus and ovary of VWD-affected pigs. VWF is known to protect FVIII from decomposition and a lack of VWF leads to lower levels of FVIII. Our results suggest that a reduced F8 expression primarily might contribute to those reduced FVIII levels in VWD-affected pigs. Additional significant results involving the pregnant pigs were detected for CALR, EDN1, and LGALS3. These genes are promising candidates for more detailed future studies.

Generation of glycans depleted decellularized porcine pericardium, using digestive enzymatic supplements and enzymatic mixtures for food industry.

BACKGROUND: Xenogeneic pericardium has been used largely for various applications in cardiovascular surgery. Nevertheless, xenogeneic pericardial patches fail mainly due to their antigenic components. The xenoantigens identified as playing a major role in recipient immune response are the Galα1-3Gal (α-Gal) epitope, the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), and the porcine SDa antigen, associated with both proteins and lipids. The reduction in glycans from porcine pericardium might hinder or reduce the immunogenicity of xenogeneic scaffolds. METHODS: Decellularized porcine pericardia were further treated at different time points and dilutions with digestive enzymatic supplements and enzymatic mixtures applied for food industry, for the removal of potentially immunogenic carbohydrates. Carbohydrates removal was investigated using up to 8 different lectin stains for the identification of N- and O-glycosylations, as well as glycolipids. Histoarchitectural changes in the ECM were assessed using Elastica van Gieson stain, whereas changes in mechanical properties were investigated via uniaxial tensile test and burst pressure test. RESULTS: Tissues after enzymatic treatments showed a dramatic decrease in lectin stainings in comparison to tissues which were only decellularized. Histological assessment revealed cell-nuclei removal after decellularization. Some of the enzymatic treatments induced elastic lamellae disruption. Tissue strength decreased after enzymatic treatment; however, treated tissues showed values of burst pressure higher than physiological transvalvular pressures. CONCLUSIONS: The application of these enzymatic treatments for tissue deglycosylation is totally novel, low cost, and appears to be very efficient for glycan removal. The immunogenic potential of treated tissues will be further investigated in subsequent studies, in vitro and in vivo.

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)-silenced islet cell clusters (ICC). Single-cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA-silenced ICCs-derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs-derived cells. Xenogeneic T cell immune responses, NK cell and antibody-mediated cellular-dependent immune responses were significantly decreased in SLA-silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single-cell engineering and post-transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.

Anticonvulsant effects after grafting of rat, porcine, and human mesencephalic neural progenitor cells into the rat subthalamic nucleus.

Cell transplantation based therapy is a promising strategy for treating intractable epilepsies. Inhibition of the subthalamic nucleus (STN) or substantia nigra pars reticulata (SNr) is a powerful experimental approach for remote control of different partial seizure types, when targeting the seizure focus is not amenable. Here, we tested the hypothesis that grafting of embryonic/fetal neural precursor cells (NPCs) from various species (rat, human, pig) into STN or SNr of adult rats induces anticonvulsant effects. To rationally refine this approach, we included NPCs derived from the medial ganglionic eminence (MGE) and ventral mesencephalon (VM), both of which are able to develop a GABAergic phenotype. All VM- and MGE-derived cells showed intense migration behavior after grafting into adult rats, developed characteristics of inhibitory interneurons, and survived at least up to 4 months after transplantation. By using the intravenous pentylenetetrazole (PTZ) seizure threshold test in adult rats, transient anticonvulsant effects were observed after bilateral grafting of NPCs derived from human and porcine VM into STN, but not after SNr injection (site-specificity). In contrast, MGE-derived NPCs did not cause anticonvulsant effects after grafting into STN or SNr (cell-specificity). Neither induction of status epilepticus by lithium-pilocarpine to induce neuronal damage prior to the PTZ test nor pretreatment of MGE cells with retinoic acid and potassium chloride to increase differentiation into GABAergic neurons could enhance anticonvulsant effectiveness of MGE cells. This is the first proof-of-principle study showing anticonvulsant effects by bilateral xenotransplantation of NPCs into the STN. Our study highlights the value of VM-derived NPCs for interneuron-based cell grafting targeting the STN.

Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties.

BACKGROUND: The programmed cell death-1 (PD-1, CD279)/PD-Ligand1 (PD-L1, CD274) receptor system is crucial for controlling the balance between immune activation and induction of tolerance via generation of inhibitory signals. Expression of PD-L1 is associated with reduced immunogenicity and renders cells and tissues to an immune-privileged/tolerogenic state. METHODS: To apply this concept for clinical xenotransplantation, we generated human (h)PD-L1 transgenic pigs and characterized expression and biological function of the transgene at the cellular level. RESULTS: The hPD-L1 was detected in kidney, heart, and pancreas. In addition, peripheral blood mononuclear cells (PBMC), cultured fibroblasts, and endothelial cells were hPD-L1 positive (hPD-L1+ ). The hPD-L1 levels were increased by the treatment of transgenic cells with human cytokines (eg, TNF-α), suggesting a regulatable mode of transgene expression. Compared to cells from wild-type pigs, hPD-L1+ PBMC had a significantly reduced capacity to stimulate proliferation of human CD4+ T cells. Moreover, fibroblasts from hPD-L1 transgenic pigs were partially protected from cell-mediated lysis by human cytotoxic effector cells. CONCLUSIONS: These data indicate a low immunogenic, immune-protected status of cells from hPD-L1 transgenic pigs. The integration of the hPD-L1 concept into existing multi-transgenic pigs is promising to achieve long-term survival of porcine xenografts in non-human primate recipients.

Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic effects. Although efficient cardioprotection after HO-1 overexpression has been reported in rodents, its role in attenuating post-ischemic inflammation is unclear. OBJECTIVES: This study assessed the efficacy of recombinant adenoassociated virus (rAAV)-encoding human heme oxygenase-1 (hHO-1) in attenuating post-ischemic inflammation in a murine and a porcine ischemia/reperfusion model. METHODS: Murine ischemia was induced by 45 min of left anterior descending occlusion, followed by 24 h of reperfusion and functional as well as fluorescent-activated cell sorting analysis. Porcine hearts were subjected to 60 min of ischemia and 24h of reperfusion before hemodynamic and histologic analyses were performed. RESULTS: Human microvascular endothelial cells transfected with hHO-1 displayed an attenuated interleukin-6 and intercellular adhesion molecule 1 expression, resulting in reduced monocytic THP-1 cell recruitment in vitro. In murine left anterior descending occlusion and reperfusion, the post-ischemic influx of CD45(+) leukocytes, Ly-6G(+) neutrophils, and Ly-6C(high) monocytes was further exacerbated in HO-1-deficient hearts and reversed by rAAV.hHO-1 treatment. Conversely, in our porcine model of ischemia, the post-ischemic influx of myeloperoxidase-positive neutrophils and CD14(+) monocytes was reduced by 49% and 87% after rAAV.hHO-1 transduction, similar to hHO-1 transgenic pigs. Functionally, rAAV.hHO-1 and hHO-1 transgenic left ventricles displayed a smaller loss of ejection fraction than control animals. CONCLUSIONS: Whereas HO-1 deficiency exacerbates post-ischemic cardiac inflammation in mice, hHO-1 gene therapy attenuates inflammation after ischemia and reperfusion in murine and porcine hearts. Regional hHO-1 gene therapy provides cardioprotection in a pre-clinical porcine ischemia/reperfusion model.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

Lack of antibody response in pigs immunized with the transmembrane envelope protein of porcine endogenous retroviruses.

Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.

Development of sensitive methods for detection of porcine endogenous retrovirus-C (PERV-C) in the genome of pigs.

Porcine endogenous retroviruses (PERV) represent a risk for xenotransplantation using pig cells, tissues or organs. PERV-A and PERV-B are present in the genome of all pigs and both infect human cells in vitro. PERV-C infects only pig cells and it is integrated in the genome of most, but not all pigs. Recombinants between PERV-A and PERV-C were described that infect human cells and replicate at high titres. To avoid such recombinations, PERV-C positive animals should not be used for breeding animals suited for xenotransplantation. In order to detect PERV-C positive pigs, different methods were developed such as specific PCRs using different primers, a highly sensitive nested PCR and a real-time PCR allowing measurement of proviral copy numbers. The real-time PCR was found to be useful to discriminate between contamination and actual provirus copies. The PCRs were optimized and their sensitivity was determined. Screening can be started with PCR1, if the result is negative, PCR2 to PCR5 or the nested PCR should be used, if the result is positive, the real-time PCR should be used to exclude contaminations. All methods were used to evaluate the prevalence of PERV-C and to identify PERV-C free animals. Due to the risk of contamination with cells from other animals testing should be performed with blood cells, not with ear biopsies.

Nuclear and nucleolar reprogramming during the first cell cycle in bovine nuclear transfer embryos.

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development.

Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

BACKGROUND: Porcine organs with transgenic expression of anti-apoptotic and anti-inflammatory genes like the human A20 gene (hA20), a tumor necrosis factor-alpha (TNF-alpha)-inducible gene, may control the acute vascular rejection (AVR) of porcine xenografts. The human A20 molecule possesses protective features against inflammatory and apoptotic stimuli in various cell types including endothelial cells, rendering it a promising candidate for transgenic pig production in the context of xenotransplantation. Here, we produced pigs transgenic for hA20 and investigated whether hA20-transgenic porcine aortic endothelial cells (PAECs) were resistant against the induction of apoptosis in vitro and to what extent hA20-transgenic porcine hearts were protected against ischemia/reperfusion (I/R) injury. METHODS: Porcine fetal fibroblasts (PFFs) were transfected with the vector pCAGGSEhA20-IRESNEO containing a chicken beta-actin/rabbit beta-globin (CAGGS)-promoter element, known to provide ubiquitous gene expression in both mice and pigs. Transfected PFFs were then used in somatic cell nuclear transfer (SCNT). Three hA20-transgenic pigs were killed for PAEC isolation and organ mRNA and protein expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern and Western Blotting. PAECs were tested for susceptibility to apoptosis after TNF-alpha challenging and triggering of the CD95(Fas)/CD95Ligand pathway. Five transgenic and three wild type animals were subjected to an I/R experiment followed by measurement of infarct size, myeloperoxidase (MPO) activity and subendocardial segmental shortening (SES) to assess protective effects of hA20 in the porcine myocardium. RESULTS: The hA20-transgenic pigs developed normally and expression of hA20 was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts and were less susceptible to the induction of cell death by CD95(Fas)L. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size did not differ between the two groups after ischemic assault, hA20-transgenic porcine hearts showed significantly lower MPO activity and better hemodynamic performance (determined as SES) than their wild type counterparts. CONCLUSIONS: The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-alpha-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies.

Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts.

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs.

BACKGROUND: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. METHODS: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. RESULTS: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. CONCLUSION: This strategy may lead to animals compatible with PERV safe xenotransplantation.

Production of viable pigs from fetal somatic stem cells.

Fetal somatic stem cells (FSSCs) are a novel type of somatic stem cells that have recently been discovered in primary fibroblast cultures from pigs and other species. The goal of the present study was to produce viable piglets from FSSCs. NT complexes were prepared from both FSSCs and porcine fetal fibroblasts (pFF) to permit comparison of these two donor cell types. FSSCs from isolated attached colonies were compared with pFF in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups, while blastocyst rates were significantly higher when using pFF as donor cells. FSSCs of three different size categories derived from dissociation of spheroids yielded similar results. The use of FSSCs of 15-20 microm in size yielded similar cleavage and blastocyst rates as fetal fibroblasts. In the final experiment NT complexes produced from FSSCs were transferred to foster mothers. After transfer to prepubertal gilts, three of seven recipients established pregnancies and delivered seven piglets, of which three piglets were viable and showed normal development. Results for the first time demonstrate that FSSCs are able to produce cloned embryos, and that pregnancies can be established and viable piglets can be produced.

Prevalence of stroke and vascular risk factors among first-degree relatives of stroke patients and control subjects. A prospective consecutive study.

BACKGROUND: Genetic and environmental factors may be of importance for stroke risk. We assessed the prevalence of stroke and vascular risk factors among first-degree relatives and spouses of stroke patients and control subjects. METHODS: As a part of the Lund Stroke Register study, we asked 925 consecutive patients with first-ever stroke and 286 control subjects to complete a questionnaire about all their first-degree relatives and spouses. The questionnaires addressed whether these relatives had been affected by stroke or TIA, hypertension, heart disease, diabetes mellitus, and if they were smokers. RESULTS: A total of 606 patients and 261 control subjects returned the questionnaire, providing information on 4,972 first-degree relatives and 738 spouses. The prevalence of stroke or TIA was 12.3% among first-degree relatives of patients and 7.5% among first-degree relatives of control subjects (OR 1.74, 95% CI 1.36-2.22). Corresponding results for hypertension were 21.0 and 16.7% (OR 1.33, 95% CI 1.10-1.60). The prevalences of heart disease, diabetes mellitus and smoking did not differ significantly between first-degree relatives of patients and control subjects. Spouses of patients and control subjects had similar prevalences of stroke or TIA and vascular risk factors. CONCLUSIONS: The prevalences of stroke or TIA and hypertension are higher among first-degree relatives of stroke patients than among first-degree relatives of control subjects. This, and the lack of differences between spouses of patients and control subjects, indicates that an increased risk of stroke may in part be explained by heritability of hypertension.

Yeast CAL1 is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing.

A 2.3-kilobase pair DNA fragment of the yeast CAL1 gene was cloned by complementation of the cal1-1 mutation, which causes a defect in nuclear division and bud formation (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1984) Mol. & Gen. Genet. 193, 389-394). Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) encoding a polypeptide of 376 amino acids. Comparative analysis of the predicted amino acid sequence has shown that the CAL1 product has similarity to two yeast proteins: the DPR1 (RAM) gene product that is involved in processing of ras protein at the farnesylation step, and the essential ORF2 protein whose structural gene has a head-to-head arrangement with PRP4, which is involved in mRNA processing. Functional homology between CAL1 and DPR1 has also been suggested from genetic evidence that multiple copies of the CAL1 gene suppress the growth defects of a dpr1 null mutant at high temperature. This suppression is Ca(2+)-dependent, since it was not observed in complete medium containing 200 microM CaCl2 but was apparent in medium containing 100 mM CaCl2. From sequence analysis of the cal1-1 mutation, together with the alignment of the three gene products, we have concluded that the conserved Gly328 in the C terminus is important for activity. We suggest that the CAL1 protein participates in a ras-like C-terminal modification of proteins involved in nuclear division and bud growth.