Gestational weight gain, appetite regulating hormones, and metformin treatment in polycystic ovary syndrome: A longitudinal, placebo-controlled study.

OBJECTIVE: To explore mechanisms that modulate gestational weight gain (GWG) in women with polycystic ovary syndrome (PCOS) and healthy controls. DESIGN: Sub-sample of randomised controlled trials (PCOS) combined with a prospective cohort (controls). SETTING: Eleven Norwegian, Swedish, and Icelandic hospitals. POPULATION: Pregnant women with PCOS treated with metformin (PCOS-M, n = 36) or placebo (PCOS-P, n = 37), and healthy pregnant women (HC, n = 15). METHODS: Serum levels of the appetite regulating hormones leptin, ghrelin, allopregnanolone, and soluble leptin receptor (sOB-R) were determined in the first and third trimesters. MAIN OUTCOME MEASURES: Excessive GWG (eGWG) relative to body mass index according to Institute of Medicine (IOM) guideline. Serum leptin/sOB-R ratio, or free-leptin-index (FLI), as biomarker of leptin sensitivity. Serum ghrelin and allopregnanolone levels. RESULTS: The overall prevalence of eGWG was 44% (38/86). Women with eGWG had higher first and third trimester FLI (P < 0.001), and lower third trimester allopregnanolone levels (P = 0.003) versus women with non-eGWG. The prevalence of eGWG was lower in PCOS-M versus PCOS-P (28% versus 62%, odds ratio = 0.4, 95% CI 0.2-0.8, P = 0.005). FLI decreased during pregnancy in PCOS-M (P = 0.01), but remained unaltered in PCOS-P and HC. Ghrelin and allopregnanolone levels were comparable in PCOS-M, PCOS-P and HC throughout pregnancy. CONCLUSION: Excessive GWG is associated with enhanced leptin resistance, and attenuated physiological increase in serum allopregnanolone levels during pregnancy. Metformin reduces the risk for eGWG and improves leptin sensitivity in pregnant women with PCOS. TWEETABLE ABSTRACT: Metformin counteracts excessive weight gain and leptin resistance in pregnant women with polycystic ovary syndrome.

Growth Restriction in the Offspring of Mothers With Polycystic Ovary Syndrome.

Importance: Polycystic ovary syndrome (PCOS) is a common endocrine disorder, characterized by subfertility, increased risk of metabolic diseases, and pregnancy complications. Previous studies diverge regarding the association between maternal PCOS and newborn anthropometrics. Objective: To explore the association between maternal PCOS and newborn anthropometrics and the modifying effects of maternal body mass index, PCOS phenotype, and gestational diabetes. Design, Setting, and Participants: This cohort study followed up women from the first half of pregnancy to birth and combined data from 3 clinical trials of pregnant women with PCOS and a reference population consisting of participants in the Norwegian Mother, Father, and Child Cohort (MoBa) Study, with data from the Medical Birth Registry of Norway. The recruitment period for the clinical trials was between October 1, 2000, and August 31, 2017, and for MoBa, between July 1, 1999, and December 31, 2008. Participants included women with singleton pregnancies and live-born children. Data were analyzed from January 1 to June 15, 2023. Exposure: Maternal PCOS status. Main Outcomes and Measures: Newborn birth weight, birth length, and head circumference as continuous variables and z scores, and ponderal index (calculated as the birth weight in grams × 100 divided by the birth length in centimeters cubed), placenta weight, and ratio of birth weight to placenta weight (BWPW). Results: The cohort included 390 pregnant women with PCOS (mean [SD] age, 29.6 [4.2] years) and 68 708 women in the reference group (mean [SD] age, 30.4 [4.5] years). Offspring in the PCOS group had lower birth weight, birth length, and head circumference than in the reference group offspring. The estimated mean differences in z scores were -0.26 (95% CI, -0.38 to -0.14) for birth weight, -0.19 (95% CI, -0.33 to -0.05) for birth length, and -0.13 (95% CI, -0.26 to -0.01) for head circumference. The PCOS group also had a lower ponderal index (-0.04 [95% CI, -0.07 to -0.004] g × 100/cm3) and placenta weight (-24 [95% CI, -43 to -5)] g), and higher BWPW ratio (0.4 [95% CI, 0.3 to 0.5]). The association between growth restriction and PCOS was more apparent when additionally adjusting for body mass index. Neither PCOS phenotype nor gestational diabetes diagnosis was associated with neonatal anthropometry in women with PCOS. Conclusions and Relevance: In this cohort of mother-infant pairs, maternal PCOS status was associated with lower birth weight, shorter birth length, and smaller head circumference in the offspring. This growth restriction was more pronounced when adjusting for BMI, providing insight into the association between PCOS and body mass index. The study contributed to the understanding of how PCOS affects the offspring.

Metformin serum concentrations during pregnancy and post partum - A clinical study in patients with polycystic ovary syndrome.

BACKGROUND AND OBJECTIVES: Metformin is used to treat gestational diabetes. It is also used to treat women with polycystic ovary syndrome and has been shown to prevent late miscarriage and preterm birth. However, increased renal clearance during pregnancy causes a decline in serum concentrations of metformin. The aim of this study was to explore the time course of the pregnancy-related changes in metformin pharmacokinetics and the return to the non-pregnant state. METHOD: A subgroup of women in the PregMet2 study (n = 73) agreed to provide serum samples at three time-points in pregnancy (gestational weeks 19, 28 and 32) and once in post partum, (either 2, 4 or 8 weeks after delivery). Serum metformin concentrations were compared using a four-parameter logistic model. FINDINGS: The mean steady-state serum concentration of metformin during pregnancy was 9.39 µmoL/L, whereas the post partum concentration was 12.36 µmoL/L, an increase of 32% (p = 0,019). This change took place already during the first 2 weeks post partum. CONCLUSION: Clinicians who treat pregnant women with metformin should be aware of the significant decrease in metformin concentration mediated by pregnancy, and the rapid increase after delivery, as it may impact both the therapeutic efficacy and the risk of adverse drug reactions.

Changes in Serum Cytokines Throughout Pregnancy in Women With Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with low-grade inflammation and increased incidence of pregnancy complications, but its influence on the maternal immune system in pregnancy is unknown. Longitudinal serum cytokine profiling is a sensitive measure of the complex immunological dynamics of pregnancy. OBJECTIVE: This work aimed to determine the immunological dynamics of serum cytokines throughout pregnancy in women with PCOS and compare it to pregnancy in women without PCOS. METHODS: A post hoc analysis was conducted of longitudinal serum samples from 2 randomized, placebo-controlled multicenter studies of pregnant women with PCOS and 2 studies of pregnant women without PCOS. Pregnant women with PCOS (n = 358) and without PCOS (n = 258, controls) provided 1752 serum samples from 4 time points in pregnancy (weeks 10, 19, 32, and 36). Main outcome measures included maternal serum levels of 22 cytokines and C-reactive protein (CRP) at 4 time points in pregnancy. RESULTS: Women with PCOS showed marked immunological changes in serum cytokines throughout pregnancy. Compared to controls, women with PCOS showed higher levels of 17 cytokines and CRP at week 10 of pregnancy and a distinct cytokine development throughout pregnancy. The immunological dynamics in women with PCOS was significantly affected by maternal body mass index, smoking, and fetal sex. CONCLUSION: Pregnancy in women with PCOS was associated with a strong early mobilization of inflammatory and other serum cytokines persisting throughout pregnancy, indicating a more activated immune status. These findings provide a novel basis for further study of PCOS and pregnancy complications.

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

Use of metformin to treat pregnant women with polycystic ovary syndrome (PregMet2): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) have an increased risk of pregnancy complications. Epi-analysis of two previous randomised controlled trials that compared metformin with placebo during pregnancy in women with PCOS showed a significant reduction in late miscarriages and preterm births in the metformin group. The aim of this third randomised trial (PregMet2) was to test the hypothesis that metformin prevents late miscarriage and preterm birth in women with PCOS. METHODS: PregMet2 was a randomised, placebo-controlled, double-blind, multicentre trial done at 14 hospitals in Norway, Sweden, and Iceland. Singleton pregnant women with PCOS aged 18-45 years were eligible for inclusion. After receiving information about the study at their first antenatal visit or from the internet, women signed up individually to participate in the study. Participants were randomly assigned (1:1) to receive metformin or placebo by computer-generated random numbers. Randomisation was in blocks of ten for each country and centre; the first block had a random size between one and ten to assure masking. Participants were assigned to receive oral metformin 500 mg twice daily or placebo during the first week of treatment, which increased to 1000 mg twice daily or placebo from week 2 until delivery. Placebo tablets and metformin tablets were identical and participants and study personnel were masked to treatment allocation. The primary outcome was the composite incidence of late miscarriage (between week 13 and week 22 and 6 days) and preterm birth (between week 23 and week 36 and 6 days), analysed in the intention-to-treat population. Secondary endpoints included the incidence of gestational diabetes, preeclampsia, pregnancy-induced hypertension, and admission of the neonate to the neonatal intensive care unit. We also did a post-hoc individual participant data analysis of pregnancy outcomes, pooling data from the two previous trials with the present study. The study was registered with ClinicalTrials.gov, number NCT01587378, and EudraCT, number 2011-002203-15. FINDINGS: The study took place between Oct 19, 2012, and Sept 1, 2017. We randomly assigned 487 women to metformin (n=244) or placebo (n=243). In the intention-to-treat analysis, our composite primary outcome of late miscarriage and preterm birth occurred in 12 (5%) of 238 women in the metformin group and 23 (10%) of 240 women in the placebo group (odds ratio [OR] 0·50, 95% CI 0·22-1·08; p=0·08). We found no significant differences for our secondary endpoints, including incidence of gestational diabetes (60 [25%] of 238 women in the metformin group vs 57 [24%] of 240 women in the placebo group; OR 1·09, 95% CI 0·69-1·66; p=0·75). We noted no substantial between-group differences in serious adverse events in either mothers or offspring, and no serious adverse events were considered drug-related by principal investigators. In the post-hoc pooled analysis of individual participant data from the present trial and two previous trials, 18 (5%) of 397 women had late miscarriage or preterm delivery in the metformin group compared with 40 (10%) of 399 women in the placebo group (OR 0·43, 95% CI 0·23-0·79; p=0·004). INTERPRETATION: In pregnant women with PCOS, metformin treatment from the late first trimester until delivery might reduce the risk of late miscarriage and preterm birth, but does not prevent gestational diabetes. FUNDING: Research Council of Norway, Novo Nordisk Foundation, St Olav's University Hospital, and Norwegian University of Science and Technology.

Quantitative analysis of mammalian chromosome by scanning transmission soft X-ray microscopy.

Soft X-ray spectromicroscopy was applied to study the quantitative distribution of DNA and protein in a mammalian chromosome at the spatial resolution of 100 nm. The quantities of DNA and protein were evaluated using 1s-π* transition in the NEXAFS spectra at the nitrogen K absorption edge. DNA was not uniformly distributed in the chromosome and DNA/protein ratio was less than 0.497. The present analysis revealed the clues to identify other molecules that contribute to the absorption spectrum of the sample. The results suggested that accumulation of the absorption spectra of relevant molecules would support the refinement of the analysis.

Monitoring of hemostatic abnormalities in major orthopedic surgery patients treated with edoxaban by APTT waveform.

INTRODUCTION: An analysis of the activated partial thromboplastin time (APTT) in major orthopedic surgery patients receiving edoxaban for the prevention of venous thromboembolism (VTE) was carried out. METHODS: The APTT waveform was analyzed in the above patients to monitor edoxaban administration. RESULTS: Of these 99 patients, 12 exhibited deep vein thrombosis, and 25 had massive bleeding. An increased biphasic pattern of the APTT waveform was observed after the administration of edoxaban, but there were no significant differences between the patients with and without complications. The peak times of acceleration, velocity, and 1/2 fibrin formation were significantly prolonged after the administration of edoxaban, especially in patients with massive bleeding, and were moderately correlated with the anti-Xa activity. While the heights of velocity and acceleration peak 2 were lower in patients receiving warfarin treatment than in those receiving edoxaban, the widths of these parameters were significantly longer. The height of 1/2 fibrin formation and the width of acceleration peaks 1 and 2 and the velocity were significantly increased after the administration of edoxaban. CONCLUSION: The peak time of the APTT waveform was significantly prolonged after the administration of edoxaban. The analysis of the APTT waveform may therefore be useful for the prediction of the risk of massive bleeding.

Linearization and integration of DNA into cells preferentially occurs at intrinsically curved regions from human LINE-1 repetitive element.

A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5' terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5' termini.

Molecular cloning and characterization of a steroid receptor-binding regulator of G-protein signaling protein cDNA.

Steroid hormone receptors are composed of six major functional domains, i.e. the A/B domains as the activation function 1 domain (AF-1), domain C as the DNA-binding domain, domain D as a hinge domain and domain E/F as the ligand-dependent transcriptional domain (AF-2). They regulate gene transcription through interactions with various nuclear factors of their domains, such as AF-1 and AF-2. We have insufficient knowledge of the function of the DNA-binding domain (domain C) except for its DNA-binding function or the hinge domain (domain D). Therefore, we attempted to identify factors interacting with the domains by using a yeast two-hybrid system. Domains C and D of estrogen receptor alpha were used as a bait to isolate cDNA clones from a rat ovary cDNA library. We isolated the cDNA clone of a novel steroid receptor-binding protein bearing the regulator of G-protein signaling (RGS) designated as SRB-RGS. The protein repressed the transcriptional activity of estrogen receptor alpha, suggesting cross-talk of steroid hormones and peptide hormones (or growth factors) for signal transductions mediated by SRB-RGS.

Cloning and recombinant expression of rat and human kynureninase.

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.

Detection of DNA cleavage in apoptotic cells.

At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice.

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.

Effect of neonatal thyroidectomy on growth hormone secretion in the rat.

The influence of neonatal thyroidectomy (Tx) on GH production was investigated by means of Northern blot analysis. Tx resulted in a significant decrease in pituitary GH mRNA levels after 10, 15 and 20 days. The changes of pituitary GH mRNA were soon reflected in pituitary GH content. There was, however, no significant difference in pituitary GH mRNA levels and GH content between Tx and sham-operated rats at 5 days old. The pituitary GH cells were significantly decreased in number 15 and 20 days after Tx. These data suggest that GH mRNA is transcribed, independent of thyroid hormone, in the rat anterior pituitary gland during early neonatal life. In addition, the present study ascertained that GH dependence on thyroid hormone is acquired between the 5th and 10th day of neonatal life.

Activation of a novel microglial gene encoding a lysosomal membrane protein in response to neuronal apoptosis.

In an attempt to understand the molecular mechanism of microglial activation in response to neuronal death or degeneration, we have employed cerebellar cell cultures prepared from P7 rats and grown in normal K(+) (5.4 mM) medium. Under this condition, glial cells respond to degeneration and cell death of granule neurons that begins to occur at 4 days in vitro (DIV). Here we describe a novel gene, granule cell death-10 (gcd-10) that is expressed in microglia and up-regulated in an early period of granule cell death. gcd-10 is homologous to the mouse lysosomal-associated multispanning membrane protein (LAPTm5) with hematopoietic origin. Immunocytochemistry and vital staining with acridine orange revealed that GCD-10 was localized at the perinuclear area of cultured microglia and COS 1 cells infected with a GCD-10-expressing adenoviral vector. In cerebellar cell cultures, however, GCD-10 was markedly up-regulated and widely distributed to the cytoplasm, which paralleled the localization of the ED1 antigen, the lysosomal marker. In vivo, gcd-10 is expressed mainly in the brain and the spleen, and was up-regulated upon nerve injury in retina 7 days after optic nerve transection. These findings suggest that gcd-10 is involved in the dynamics of lysosomal membranes associated with microglial activation both in vitro and in vivo.

Vascular surgical society of great britain and ireland: thrombelastography can differentiate ischaemic from haemorrhagic stroke

BACKGROUND: Thrombolysis could be a major step forward in the treatment of acute ischaemic stroke. Early treatment is essential to maximize therapeutic benefit. Imaging by computed tomography (CT) or magnetic resonance imaging is mandatory to exclude haemorrhagic stroke before initiation of therapy. Thrombelastography (TEG), a test of global haemostasis, produces a characteristic tracing over 15-30 min. The potential of TEG to differentiate patients with ischaemic from haemorrhagic stroke was investigated. METHODS: Fifteen patients with a clinical diagnosis of acute stroke were studied. Fibrinogen levels and lipid profiles were measured, and CT of the brain, coagulation screens and TEG were performed. The CT scans were interpreted by a single radiologist. TEG data were classified as normal (index less than 2), hypercoagulable (index 2-3) or profoundly hypercoagulable (index greater than 3). RESULTS: There was no correlation between any of the parameters studied other than TEG with CT findings. Both patients with a haemorrhagic stroke showed normal findings on TEG. Eleven of the 12 patients with ischaemic stroke were hypercoagulable or profoundly hypercoagulable. CONCLUSION: TEG is capable of differentiating haemorrhagic from ischaemic stroke. It has the potential to target thrombolytic therapy for those patients most likely to benefit.

Immobilization stress reduced the expression of neurotrophins and their receptors in the rat brain.

Exposure to stressful events and elevated level of stress hormones are associated with impaired spatial memory and neuronal damage in the hippocampus. These neurons are considered to be maintained by neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and trk family of neurotrophin receptors. Male Wistar rats (6 weeks old) were exposed to immobilization stress for 8 h and their brains were processed for in situ hybridization histochemistry. Exposure to long-lasting immobilization stress reduced mRNA levels for neurotrophins and their high affinity receptors in the brain, especially in the hippocampus. Our results provide, some new information that may be relevant to the pathogenesis of stress-induced disturbances of memory and learning.

Intermediate lymphocytic lymphoma with both t(14;19)(q32.3;q13.1) and t(3;22)(q27;q11.2).

Chromosome translocations involving various chromosomes sites, including the sites of immunoglobulin loci (14q32,2p12,22q11) represent recurrent aberration in non-Hodgkin lymphoma (NHL). We report a novel case of intermediate lymphocytic lymphoma (ILL) with both t(14;19)(q32.3;q13.1) and t(3;22)(q27;q11.2). The t(14;19)(q32.3;q13.1) and t(3;22) (q27;q11.2) may represent reciprocal recombinations between immunoglobulin (Ig) H chain gene (14q32.3) and bcl-3(19q13.1) and between Ig lambda chain gene (22q11.2) and bcl-6 (3q27), respectively.

High potassium promotes differentiation of retinal neurons but does not favor rod differentiation.

Neural retinal cells of newborn rats were cultured under dissociated culture conditions. Differentiation of several types of retinal cells was confirmed by immunohistochemical detection of type-specific neural phenotypes. We used Thy-1.1 antigen as a ganglion cell marker, HPC-1 or GABA as an amacrine cell marker and rhodopsin as a rod cell marker. With a high concentration of potassium (38 mM), expression of the respective neural phenotypes were differentially affected. High K+ increased the number of Thy-1.1 positive cells 6 to 8 fold, and drastically promoted their neurite extension. The same culture conditions, however, reduced considerably the number of rhodopsin positive cells, possibly due to the unique membrane properties of photoreceptors. A high K+ concentration also promoted differentiation of HPC-1 positive and GABA positive cells, but to a lesser extent than the Thy-1.1 positive cells. Several possibilities were examined to understand the effect of a high K+ concentration on retinal neural cells. The total cell number in cultures with a high K+ concentration was approximately half of that in control cultures at day 3 and slightly smaller at day 11, suggesting that high K+ did not have a positive general effect on the proliferation or survival of retinal cells. Naturally occurring neuronal death (apoptosis) is a well-known phenomenon during retinal development. A histochemical method for detecting DNA fragmentation, a step preceding apoptosis, showed that high K+ had no preventive effect. BrdU (bromodeoxyuridine) immunohistochemistry showed that high K+ did not seem to enhance proliferation of neural precursor cells. These results indicate that a high K+ concentration promotes the expression of neuronal phenotypes but is not a favorable condition for rod differentiation. Since a high K+ concentration is considered to induce depolarization of nerve cells, the present results suggest an anterograde influence from surrounding neuronal cells, through chronic depolarization by elevated K+, is essential for the differentiation and maturation of retinal cells.

Heat-induced apoptosis and p53 in cultured mammalian cells.

Heat-induced apoptosis was studied in M10 and MOLT-4 cells by determining nuclear morphological changes, decrease in cell size, DNA degradation into fragments of about 30 kbp, and the appearance of internucleosomal DNA fragments (DNA ladders). Morphological changes in the nucleus were detected within 30 min after heat-treatment at 44 degrees C in M10 cells, but much later (> 5 h) in MOLT-4 cells. In M10 cells, 30 kbp-DNA fragments were observed even at the end of the heat-treatment and decreased 10 min later, while the DNA ladder increased at 10-30 min after heat treatment. DNA fragments of 30 kbp appeared in MOLT-4 cells at 1 h after the heat-treatment and apparently accumulated for up to 24 h. Heat-treatment increased p53 protein in MOLT-4 cells but not in M10 cells. Analysis of the DNA sequence of the p53 gene revealed that M10 cells have a heterozygous mutation in codon 173 of exon 5. These results suggest that apoptosis is induced by hyperthermia in a cell-line dependent manner, that the formation of 30 kbp-DNA fragments is a very early event in apoptosis, that DNA fragmentation into a DNA ladder occurs via the 30 kbp fragments, and that apoptosis in heat-treated M10 cells may be independent of p53.