The resonance nature of the dependence of epithelial lesions of the small intestine in mice on the interval between injections of an S-phase-specific agent--hydroxyurea

The dependence of the injury of murine small intestinal epithelium on the interval between multiple regular injections of hydroxyurea (HU) was investigated. Mice were injected 8 times with HU (5 mg per injection) in different experimental groups of animals, and the interval between injections varied from 6 to 19 hours. With the intervals between the injections close to 8 or 16.5 hours the resonance decrease of the injury was observed whereas the intervals of 6, 12 and 19 hours corresponded to maximum injury.     

Resonance nature of the relation of mouse survival to the interval between administrations of the S-specific agent--hydroxyurea

The dependence of murine survival on the intervals between periodic hydroxyurea (HU) injections was studied. The single dose of HU comprised 250 mg per kg. Intervals between injections varied from 5 to 19 hours while their number changed from 6 to 9 in different experiments. A resonant increase in the survival was observed under HU administered every 8-9 or 16 1/2 hours.     

Hindlimb unloading depresses corneal epithelial wound healing in mice.

C57BL/6 mice were subjected to hindlimb unloading (HU) for a period of 3 wk to determine the possible effects on epithelial wound healing. A standardized corneal epithelial wound was performed, and parameters of the inflammatory response and reepithelialization were analyzed over an observation period of 96 h. Wound closure was significantly retarded in mice during HU with reepithelialization being delayed by approximately 12 h. Both epithelial migration and cell division were significantly depressed and delayed. The inflammatory response to epithelial wounding was also significantly altered during HU. Neutrophils, as detected by the Gr-1 marker, were initially elevated above normal levels before wounding and during the first few hours afterward, but there was a significant reduction in neutrophil response to wounding at times where neutrophil influx and migration in controls were vigorous. A similar pattern was seen with CD11b+CD11c+ cells (monocyte lineage). Langerhans cells are normally resident within the peripheral corneal epithelium. They respond to injury by initially leaving the epithelial site within 6 h and returning to normal levels by 96 h, 2 days after reepithelialization is complete. During HU, this pattern is distinctly different, with Langerhans cell numbers slowly diminishing, reaching a nadir at 96 h, which is significantly below normal. Evidence for systemic effects of HU is provided by findings that collagen deposition within subcutaneous sponges was significantly reduced during HU. In conclusion, HU, a ground-based model simulating some physiological aspects of spaceflight, impairs wound repair of corneas. Multiple factors, both local and systemic, likely contribute to this delayed wound healing.     

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G₂ chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.     

Deoxyribonucleoside triphosphate pools in regenerating rat liver: effect of hydroxyurea and exogenous deoxypyrimidines

Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals.     

Recovery kinetics of epidermal cell proliferation after two intraperitoneal injections of epidermal extracts (chalone)

The cell kinetic effect of two intraperitoneal (IP) injections of 5 mg of crude lyophilized skin extracts given 12 h apart was assessed during the recovery period (5 to 52 h after last injection) by measuring epidermal labelling indices and the specific activity after tritiated thymidine (3HTdR) injection, and by determining the cell cycle phase distribution by flow cytometry. The skin extracts produced an epidermal chalone effect and inhibited both DNA synthesis and mitosis. A slow recovery took place from 5 to between 22 and 36 h after the last chalone injection. During this period the cell flux into DNA-synthesis and mitosis slowly recovered, but the exits were blocked and cells accumulated in the respective phases. The fluxes out opened up at the S phase about 22 h, and at the M phase about 30 h after the second chalone injection. A secondary inhibitory effect was observed at about 40 h, followed by a subsequent recovery to normal at 52 h. The similarity between the recovery kinetics after chalone and that observed after hydroxyurea (HU) is emphasised.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

The effect of hydroxyurea on the phosphorylation of zidovudine and lamivudine in human umbilical vein endothelial cells

Summary  The purpose of this study was to characterize the activation of zidovudine (ZDV) and lamivudine (3TC) in human umbilical vein endothelial cells (HUVEC) with and without hydroxyurea (HU) pretreatment. HUVEC were pretreated with HU or control media for 24 h and then incubated for an additional 3 h with ZDV or 3TC. Intracellular concentrations of parent drugs and the phosphorylated forms were determined by high-performance liquid chromatography. Pretreatment with HU resulted in more than a threefold increase in intracellular concentrations of total ZDV, with the major intracellular form being the monophosphate (>80%). The relative percentage of each ZDV form was similar between control and HU-treated cells. On the other hand, intracellular concentrations of total 3TC increased only slightly (14%) with HU pretreatment. Although the parent drug remained the major intracellular form of 3TC, there was nearly a 400% increase in the 3TC triphosphate after HU pretreatment. These data demonstrate that HU causes a large increase in the intracellular accumulation of total ZDV, whereas it increases total 3TC only slightly but improves its triphosphorylation. Given the increase in intracellular concentrations of ZDV monophosphate after HU pretreatment and that the monophosphate has no antiviral activity but is associated with toxicity, the use of HU is not a good strategy to improve ZDV activation in human endothelium. There is improved production of the active antiviral 3TC triphosphate with HU pretreatment. The combination may be beneficial in treating potential sanctuary sites such as endothelium.     

Hydroxyurea synchronization of bovine fetal spleen cells

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Hydroxyurea synchoronization of bovine fetal spleen cells.

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Effects of hydroxyurea on the mitotic activity and the G2 phase in hairless mouse epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid at various times after HU. The animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G2 was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G2 for the first 6 hr after HU injection, then the G2 compartment was emptied. The results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G2 and M. The cell kinetic effects of HU thus seem to be very complex.     

Hydroxyurea Use and Hospitalization Trends in a Comprehensive Pediatric Sickle Cell Program

Background

A decline in hospitalizations and pain episodes among those with sickle cell disease (SCD) who take hydroxyurea (HU) has been shown when compared to pre-HU patterns but paradoxically, when compared to those who have never been treated, HU recipients often have more frequent hospitalizations. This analysis evaluates the impact of increasing usage of HU on trends in hospitalizations and blood transfusions within a large SCD treatment program.

Methods

Eligibility was restricted to patients with Hb SS or Hb Sβ⁰-thalassemia who were 2–18 years old between 2006–2010 and received care at St. Jude Children''s Research Hospital (N = 508). Hospitalizations and blood transfusions were calculated for each of the years under study for those exposed and never exposed to HU. Differences in number of hospitalizations before and after HU initiation were compared.

Results

The proportion of patients receiving HU increased by 4% per year on average. In the HU exposed group, a modest decline in mean per-patient hospitalizations and per-patient hospital days occurred, while those never exposed to HU trended toward a slight increase over time. Rates of blood transfusions declined among those on HU but not in patients never exposed to HU. Patients on HU had a median of one fewer hospital admission in the year after initiation of HU, compared to the year prior. Two deaths occurred in the patient population, both of whom were not exposed to HU.

Conclusions

Increasing usage of HU was concurrent with decreased hospitalization rates and blood transfusions. Our results support the utility of HU on decreasing hospitalizations and transfusions for patients with SCD outside of the clinical trial setting.     

Effects of diet type on establishment of pregnancy and embryo development in beef heifers

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18-24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5kg concentrates and 250g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250g feed grade urea and 50g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR(?), InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15mg luprostiol was given 12h before pessary withdrawal. They received 144mg pFSH (Folltropin(?)-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1±0.5, 4.9±0.3 and 3.2±0.1mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2±0.8, 5.8±1.5 and 6.8±1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7±0.6, 3.4±1.1 and 4.8±0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4±0.8 compared with 1.0±0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0±13.3, 86.9±7.2 and 76.5±7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3±11.8, 92.0±3.5 and 86.8±5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.     

Plasma LH concentrations after single or double injections of synthetic LH-RH in dairy cows.

Milked Friesian dairy cows were given 1 or 2 i.v. injections of synthetic LH-RH. The second of 2 injections of LH-RH given 1.5 h apart induced a significantly greater release of LH than did the first. The response to this injection was also significantly greater than that caused by injections given 6 h apart. There was no significant difference between the LH responses to the two injections given 6 h apart. The total LH release produced by the two injections of 100 microgram synthetic LH-RH given 1.5 h apart was significantly greater than that produced by single injection of 200 microgram synthetic LH-RH. It is concluded that there is a short-term increase in the responsiveness of the bovine pituitary gland to LH-RH after an initial LH-RH stimulus.     

Quantification of the Effects of Resperine on Gonadotroph Expression in the Pituitary of Goldfish (Carassius auratus)

In many teleosts, the control of gonadotropin II (or luteinizing hormone) secretion is under the dual control of stimulatory and inhibitory neuroendocrine factors. The principal stimulating factor is gonadotropin-releasing hormone and the main inhibitor is dopamine. Inhibiting the activities of dopamine by antidopaminergic drugs potentiates the actions of exogenous gonadotropin-releasing hormone analogs, resulting in a surge release of luteinizing hormone and ovulation and spawning in a number of different species. As the effects of blocking the inhibitory actions of dopamine on gonadotroph cytology have not been studied, goldfish were treated with 2, 4, 6 or 8 injections of reserpine (0.1 mg/kg body weight), at 48 h intervals, and the numbers of gonadotrophic cells studied at 48 h following last injection. After two injections, the number of gonadotrophic cells increased by 189% over controls; after four injections the increase was 234%; after six injections the increase was 259% and after eight injections, 288%. The results suggest that dopamine has an inhibitory influence on the numbers of gonadotrophs.     

A sequential analysis of meiosis in the male mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment

A method is described to restrict the spermatocyte population in mice and other rodents using hydroxyurea (HU) and triaziquone (T). HU affects cells in S-phase, whereas T is an agent especially active on spermatogonia and not on spermatocytes. An application of three i.p. HU injections with 12 h intervals, followed about nine days later by one i.p. T injection creates two large gaps in the spermatogenic line. The two gaps enclose a small, well-defined group of primary spermatocytes in meiotic interphase. — The development of the restricted spermatocyte population is followed day by day. The analysis of meiosis in male mice has revealed the correct sequence of meiotic, and especially prophase I stages. On account of clearly visible differences in chromosome morphology the diplotene stage could be divided into three periods. It is suggested to use the following nomenclature: pre-diffuse diplotene, diffuse diplotene and post-diffuse diplotene. The experiment was also informative about the timing of the stages in spermatocyte development by correlating the days at which the successive stages were observed with the corresponding stage of the epithelial cycle. The calculation of the position and duration of the diffuse diplotene, enables us to put forward a proposal about the significance of the diffuse diplotene. — A combination of the HU/T method with cell separation techniques provides good perspectives for detailed biochemical studies on processes taking place during meiosis.     

A technique for determining the proportion of the clonogenic cells in S phase in ENT6 cell cultures and tumors.

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (3H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by 3H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of 3H-TdR. Under these conditions, either 3H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or 3H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by 3H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogeneic tumor cells in S phase. Incubation of tumor cell suspensions with 3H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of 3H-TdR during the incubation for colony formation.     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

An improved method for the cell cycle synchronization of <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of ³H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.