Search

To assess whether neuropsychological tests and MRI measures could be used as predictors of short-term disease evolution in a population of patients with benign multiple sclerosis (B-MS).

The definition of benign multiple sclerosis (B-MS) is still controversial. This mainly takes into account the subject’s motor ability, with little or no relevance to other important features such as cognition. Moreover, no paraclinical markers are currently available to reliably identify patients who will remain benign in the long term.

To assess neocortical changes and their relevance to cognitive impairment in early relapsing–remitting (RR) multiple sclerosis (MS).

In this study the solubility to α-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time ± SE = 24.1 ± 0.9 min, n = 28; and class II, 46.7 ± 2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3 ± 3.4 min, n = 18; ICSI: 48.9 ± 2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0 ± 2.7 min, n = 13; unfertilised ICSI: 43.0 ± 2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5 ± 2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

In the mouse embryo, at approximately 11.5 days postcoitum (dpc), cells migrate from the mesonephros into the developing testis to contribute to the somatic population of the interstitial compartment (i.e., peritubular myoid cells, Leydig cells, and endothelial cells). Studies from this laboratory have shown that the interstitial population of mesenchymal cells in fetal and newborn mouse testis express the p75 neurotrophin receptor (p75NTR, formerly known as the low-affinity nerve growth factor receptor); part of the cell population progressively congregates around testis cords, later to be replaced by contractile peritubular myoid cells, which express smooth muscle cell markers. In the present study, we show that the migrating cells and the p75NTR-expressing cells are the same population. We also show that the neurotrophin receptor is a useful endogenous marker to follow cell migration within the urogenital ridge and to identify and isolate mesenchymal precursors of myoid cells. A time-course immunolocalization study of the location of p75NTR-bearing cells within the urogenital ridge of mouse embryos between 10.5 and 12.5 dpc showed that the interstitium of the fetal testis was progressively occupied by p75NTR(+) cells. The progressive increase of p75NTR expression within the developing testis was confirmed by immunoblot analysis of proteins isolated from the fetal gonads. Organ cultures of isolated testes or testis-mesonephros grafts confirmed that p75NTR(+) cells do not appear in the testis unless a mesonephros is attached to it. Cells bearing the p75NTR receptor, purified from 12.5-dpc male mouse mesonephroi by immunomagnetic sorting, were able to differentiate in vitro into myoid cells. Immunofluorescence analysis of postnatal testis sections confirmed the presence around the tubules of cells coexpressing p75NTR and α-smooth muscle actin. The ability to identify and purify precursors of myoid cells may be of considerable help for studying the mechanisms regulating their differentiation.

Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express α-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.

Several aromatic polyamides were prepared from 4,4′-[sulfonyl bis(p-benzoyl)(p-phenyleneoxy)]dibenzoic acid with various diamines by the phosphorylation route. The polymers were characterized by elemental analysis, thermogravimetric analysis, differential scanning calorimetry and infra-red analysis. The polyamides, obtained in quantitative yield, possessed inherent viscosities in the range 0.3–1 dl g−1and 10% weight loss in nitrogen and air were above 460 and 440°C, respectively. Most of the polymers were soluble in aprotic solvents. The effect of the structure on properties such as solubility and thermal behaviour were also studied.

As the effectiveness of continuous azathioprine therapy in multiple sclerosis (MS) is still controversial, we have conducted a prospective trial of the drug (2 mg/kg daily for at least 2 years) in patients with clinically definite MS, ie with remitting, remitting-progressive and progressive MS. At the end of an average 3-year follow-up 65% of the 40 patients with remitting-progressi had the same EDSS score as they had to start with. This was observed mainly in patients with longstanding MS and a high baseline EDSS. Of the 22 patients with a remitting course 77% showed no clinical deterioration and the annual relapse rate declined, although not significantly. In only one case did the remitting disease become remitting-progressive. Azathioprine would seem to have some efficacy in delaying the progression of remitting MS.

14 patients with chronic progressive multiple sclerosis, selected in a preliminary uncontrolled trial, were given a short course of intensive cyclophosphamide therapy, which was discontinued when the leukocyte count fell to 3000 cu.mm. 5 patients dropped out because of severe side effects. At 1 year follow-up were neurologically unchanged since admission to the trial; 4 remained stable at 2 years. The lack of clinical improvement, the high frequency of side effects and the proven oncogenicity of cyclophosphamide led us to discontinue the trial.

Neurotransmitter receptors have been studied by conventional electrophysiological techniques in the membrane of human ovarian oocytes isolated from ovarian fragments obtained from pre‐menopausal women undergoing abdominal surgery for gynaecological conditions. Ovarian oocytes respond to acetylcholine (ACh) concentrations as low as 10(‐10) M by hyperpolarizing the membrane and by concomitantly increasing input resistance, in a dose‐dependent manner. The response lasts as long as the transmitter is present in the extracellular fluid. No response is elicited by ionophoretically applied ACh. The ACh response has an apparent latency of less than 1 s and a reversal potential of about ‐12 mV. The response to ACh (10(‐8) ‐ 10(‐3) M) is unaffected by curare (10(‐5) ‐ 10(‐4) g/ml) and is blocked by atropine (10(‐6) ‐ 10(‐4) g/ml). This indicates that ACh receptors in the human oocyte membrane are probably muscarinic in nature. No response is elicited by the amino acids glutamate, aspartate and glycine (up to 10(‐3) M), or by noradrenaline, adrenaline and 5‐hydroxytryptamine (up to 10(‐3) M). On the basis of analogies to the response elicited by agents which activate parthenogenetic development in the oocytes of other mammals, it is suggested that the sperm‐carried ACh might be involved in activation processes triggered by sperm‐egg interaction.

The results of a bio-climatic study regarding the Sicilian territory is given. On the basis of thermometric and pluviometric data, some indexes proposed by Rivas-Martinez have been produced for 61 localities of the island. In particular, the indexes utilized for the bio-climatic classification are the following: It (index of thermicity), Iov (ombro-thermic index) and Iovc (compensated ombro-thermic index). Besides, for the individuation and delimitation of the bio-climatic belts and relative thermo-types and ombro-types other data, as average annual temperatures, average annual precipitation, geomorphology and relative climax vegetation, have been considered. On the whole, the bio-climatic belts occurring in Sicily are: inframediterranean, thermomediterranean, mesomediterranean, supramediterranean, oromediterranean and crioromediterranean. The correlations between the vegetation and bio-climate are examined too.

Integrins are a family of cell surface receptors that mediate cell-cell and cell-matrix interactions in a variety of different cellular systems. Here we show that unfertilized mouse oocytes express beta 1 class integrins both at mRNA and protein levels. Using the reverse transcription polymerase chain reaction and oligonucleotide primers based on the DNA sequence of mouse integrins, the RNA transcripts for the beta 1, alpha 5 and alpha 6 subunits were detected in unfertilized oocytes. The expression of the mRNAs is paralleled by the expression of the corresponding proteins, in fact, the alpha 5/beta 1 and the alpha 6/beta 1 complexes can be immunoprecipitated with specific antibodies form 125I-surface-labeled oocytes. Using subunit-specific antibodies we also demonstrate the presence of the alpha 3/beta 1 at the oocyte surface but alpha 1, alpha 2, alpha 4 or alpha V subunits were not detectable. Since the mouse alpha 3 DNA sequence is not available, we have not tested for the corresponding transcript. Integrin subunits alpha 6 and beta 1 were differently distributed on the oocyte surface, as visualized by immunofluorescence staining and by immunoelectron microscopy. alpha 6 antigen was mainly confined to the microvillous area of the oocyte surface, while beta 1 was more homogeneously distributed over the whole oolemma. These data demonstrate for the first time the expression of three beta 1 integrin complexes in unfertilized mouse oocytes. Such proteins may have a role in sperm-egg interaction or during very early steps of embryogenesis.

Summary.The temporal changes of metabolic coupling between the mouse oocyte and the cumulus cells which follow hCG injection in vivoand FSH treatment in vitrowere studied by measuring what fraction of [3H]uridine taken up by cumulus cells was transferred to the oocyte. Meiotic resumption and a partial coupling loss (to 35% of the initial value) spontaneously occurred in cumuli cultured in control medium. The addition of 1 μg FSH/ml in vitro, or the injection of hCG in vivocaused a delay of about 3 h in both phenomena and a near total uncoupling, together with cumulus expansion. FSH caused uncoupling even if cumulus expansion was prevented by the addition of heparin. The presence of 2 mm-dcAMP prevented meiotic resumption in cumulusenclosed oocytes and maintained a high level of co-operation for at least 6 h. The slow uncoupling observed at later times was due to cumulus expansion, because it was totally prevented by heparin. We suggest that metabolic co-operation with the cumulus oophorus and meiotic resumption are both regulated by FSH through variations of intracellular levels of cAMP.

Abstract: The polycondensation reactions between 4,4′-[sulphonyl bis(p-benzoyl)(p-phenyleneoxy)]dibenzoic acid (I) andp-phenylenediamine (II), 1,5-diaminonaphthalene (III), 4,4′-sulphonyldianiline (IV), 4,4′-diaminodiphenylsulphide (V) 4,4′-methylenedianiline (VI) and 4,4′-oxydianiline (VII) to form aromatic polyamides containing sulphone, ether and ketone linkages were attempted by a solid-solid interaction route. A stoichiometric 1∶1 molar ratio of solid reactants was dynamically heated directly in a TG/DSC apparatus, and simultaneous TG/FT-IR was performed to interpret the mechanism of reaction. The results suggest that the polycondensation is dependent on the diamine used. The formation of polyamide was successful when I interacted with II, III, VI and VII. The interaction with IV and V was in part successful because partial decarboxylation of the diacid, made unstable by the diamine, occurred before the condensation reaction.

Solid Co(II) and Ni(II) phenoxyacetates were studied by DSC and TG techniques. The results showed that they were dihydrate and tetrahydrate respectively in the solid state. No evidence of the location of water molecules in different coordination shells was obtained. Both compounds decomposed with the formation of oxide as final product; the corresponding activation energies suggested a stronger metal-carboxylate bond in the nickel salt. Mittels DSC und TG wurden Co(II)- und Ni(II)-phenoxyacetat als Feststoffe untersucht. Die Ergebnisse zeigten, daß es sich dabei im festen Zustand um das Di- bzw. Tetrahydrat handelt. Für die Lokalisation der Wassermoleküle in den verschiedenen Koordinationssphären konnten keine Beweise erbracht werden. Beide Verbindungen zersetzen sich unter der Bildung des Oxides als Endprodukt; die entsprechenden Aktivierungsenergien scheinen auf eine stärkere Metall-Carboxylat-Bindung im Nickelsalz schließen zu lassen.

The thermal behaviour of solid zinc phenoxyacetate was investigated by DSC and TG techniques. The results obtained showed that this compound is a dihydrate and that the water molecules are differently bonded. The compound melts and then decomposes in two steps, with the formation of ZnCO3 and ZnO, respectively. The thermodynamic parameters associated with the thermal processes were evaluated.

The solid-state syntheses of complexes of uranyl acetate dihydrate andN-phenylthiourea have been attempted by heating various stoichiometric mixtures of the reactants directly in a DSC and in a TA apparatus. Both the DSC and the TG results indicate that only the 1∶1 adduct is formed, independently of the molar ratios of the reactants. It appears that the reaction is complete only with a large excess ofN-phenylthiourea, in agreement with IR data.

The thermal behaviour of mixed complexes containing transition metals, α-nitroketones and pyridine or ethanol was investigated by thermogravimetric analysis (TG), differential thermal analysis (DTA) and differential scanning calorimetry (DSC). The values of the activation energies and the enthalpies for the dissociation processes have been calculated in high vacuum and isothermal conditions. Comments are made on the obtained heats and correlations with structural characteristics are looked for.

During migration and for about 2 days after their arrival in the gonadal ridges, primordial germ cells (the embryonic precursors of gametes of the adult animal) proliferate actively. Certain growth factors, such as stem cell factor and leukemia inhibitory factor, seem to be essential for survival, proliferation and possibly differentiation of mouse primordial germ cell in vivo and/or in vitro. Similarly, increase in intracellular cAMP is followed by a marked enhancement of primordial germ cell proliferation, at least in culture. In the present study, we show that pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38), two neuropeptides of the secretin-glucagon-vasoactive intestinal polypeptide-GH-releasing hormone family, stimulate in vitro proliferation of mouse primordial germ cells, bind to primordial germ cells and gonadal somatic cells (possibly to type I PACAP receptor) and activate adenylate cyclase in the same cells. Moreover, PACAP-like immunoreactivity was found in gonadal ridges, mostly on germ cell surface. In conclusion, evidence is provided that PGC proliferation can be stimulated by certain bioactive polypeptides, thus suggesting a novel regulatory role for such compounds in early gonad development.

Summary.When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm–DNA association kinetics were rapid (maximum values were reached within 20–40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.Keywords:spermatozoa; DNA; heparin; transfection; transgenic animals

The solid-state syntheses of uranyl acetate adducts with thiourea were attempted directly in a DSC apparatus according to the scheme: UO2Ac2·2 H2O(s)+n thiourea(s) → → UO2Ac2·thiourean(s)+2 H2O(v) (n=1–6), and the correspondingδH values were evaluated. At all stoichiometric ratios the same UO2Ac2·thÌourea product was obtained, to which a pentagonal bipyramidal structure was attributed. Thermoanalytical (TG and DTG) and infrared spectra measurements were also performed and agree with the DSC results.

Expansion of the cumulus cell-oocyte complex (COC) in the preovulatory mammalian follicle requires a transient induction of hyaluronan (HA) synthesis by the cumulus cells. We studied the interactions of known factors that regulate this process by isolating compact COCs from mice and inducing their expansion in vitro. Maximum HA synthesis requires either follicle-stimulating hormone (FSH) or epidermal growth factor (EGF) in combination with either a soluble factor(s) produced by the oocyte or transforming growth factor beta1. FSH (or EGF) exerts its effects during the first 2 h of incubation, before HA synthesis actually begins. The oocyte factor(s) (or transforming growth factor beta1) exerts its effects from 2 h onwards and must be continuously present throughout the subsequent approximately 10 h to achieve a maximum level of HA synthesis. FSH stimulates intracellular cAMP synthesis, which correlates with net HA production up to approximately 14 fmol/COC at 5 ng/ml FSH; however, higher concentrations of FSH increase cAMP levels approximately 10-fold higher with no additional effect on HA synthesis. EGF at saturating concentrations for HA synthesis does not stimulate cAMP above basal levels. Tyrosine kinase inhibitors genistein and tyrphostin AG18 nearly abolish the HA synthesis response to EGF and inhibit the response to FSH by approximately 60%, suggesting that a tyrosine kinase activity is involved for both factors, whereas FSH also operates partially through another signaling pathway. Actinomycin D abolishes HA synthesis if added at the beginning of culture and reduces HA synthesis by approximately 50% if added between 6-12 h when HA synthesis is normally maximal. The results suggest that regulation of HA synthesis is primarily controlled at the transcriptional level.

The ability of terephthalic acid or terephthaloyl chloride to interact in the solid phase with DL-α-alanine to form oligomeric products was studied by means of DSC and TG/FT-IR techniques. The results suggest that the polycondensation is mainly dependent on the thermal stability of the dicarboxylie acid or its derivative with respect to the aminoacid. No interaction occurs between the less thermally stable aminoacid and the dicarboxylic acid, confirming the unreactivity of the amino group towards the COOH groups. In contrast, the oligomeric species (with n=9) was obtained by interaction of DL-α-alanine with melted terephthaloyl chloride and the reaction mechanism was elucidated by means of ESC A measurements.

The sensitivity of early preimplantation mouse embryos to drugs which disrupt microfilament function (cytochalasin B-CB and cytochalasin D-CD) and microtubule assembly (colchicine, colcemid, vinblastine and griseofulvin) was examined. CD inhibited cleavage at a concentration 35-fold lower than CB (3 × 10–7 M v. 1 × 10−5 M). Treatment of 2-cell embryos for 6 h with 1 × 10−5 M CB or 1 × 10−6 M CD or continuous exposure to lower concentrations of CB or CD did not affect development to the blastocyst stage in vitro. Vinblastine inhibited cleavage at a concentration tenfold lower than colcemid or colchicine (1 × 10−8 M v. l × 10−7 M). The continuous presence of colcemid at 10−8 M did not affect the development of 2-cell embryos to the blastocyst stage, but development was reduced with vinblastine at 1 × 10−9 M and completely inhibited with colchicine at 1 × 10−8 M. The drugs produced similar responses when 2-cell embryos were treated for 6 h with concentrations that inhibited cleavage. Complete inhibition of cleavage was obtained after only a 2 h exposure to 2 × 10−7 M colchicine. A similar concentration of lumicolchicine did not affect cleavage or blastocyst formation. Embryos were less sensitive to griseofulvin; the first cleavage division was unaffected by concentrations as high as 3 × 10− 4M and only 50% of 2-cell embryos failed to cleave in 1 × 10 1 and 3 × 10 −4M griseofulvin. At these concentrations a small proportion of 1-cell embryos and the majority of the 2-cell embryos showed unequal cytoplasmic division probably caused by the formation of multipolar spindles. The continuous exposure of 2-cell embryos to 3 × 10−5 M griseofulvin did not affect blastocyst formation.

Recently ovulated mouse oocytes at the Metaphase II stage undergo parthenogenetic activation (as indicated by the formation of pronuclei) when incubated for 6 h in the presence of cycloheximide or puromycin; the activation response increases progressively with the concentration of inhibitor. Activation is induced with concentrations of cycloheximide that depress protein synthesis by more than 70%. Pronoculear formation occurs when protein synthesis is almost totally inhibited. Incubation of oocyte in Actinomycin D failed to initiate activation. The results show that the Metaphase II oocyte of the mouse synthesizes protein factor(s) which are necessary for the maintenance of the meiotic block. Other protein(s) having opposite effects and a different rate of turnover may also participate in activation since when the oocytes are treated with a high concentration of cycloheximide (10 μg ml−1) for varying periods of time, or with varying concentrations for a short period of time (1 h), a more complex activation response curve is obtained. Oocytes activated with cycloheximide are capable of further development, following transfer to the oviducts of pseudopregnant recipients, in a proportion similar to that of oocytes activated in other ways.

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 μg/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 ± 0.2 to 169 ± 13 fmol) and in the enclosed oocyte (from 0.9 ± 0.2 to 2.4 ± 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 μg/m1 FSH plus 1 mM isobutyl-1-methylxanthine (IBMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.

The thermal decomposition of CaC2O4 · H2O is studied in air and in a nitrogen atmosphere. The influence of the environment on the DTA curves is discussed. The activation energies and the frequency factors of the three reactions occurring in this decomposition have been determined by application of Kissinger's equations to the DTG curves and, for comparison, to the simultaneous DTA curves. DTG, unlike DTA, may be applied to a reaction: solid A=solid B + gas C when another transformation involving only enthalpy change is also occurring at the same time.

Institutes of Histology and General Embryology of the University of Rome and of the School of Medicine of L'Aquila, Italy(Received 5th August 1974)Microdensitometric studies of DNA content in Feulgen-stained mouse zygotes (Alfert, 1950) have shown that the pronuclei duplicate their DNA before the prophase of the first cleavage division. The earliest incorporation of labelled adenine into DNA was autoradiographically detected by Sirlin & Edwards (1959) 13 hr after ovulation; according to Mintz (1965), DNA synthesis takes place by the end of the first third of the cycle. In the present study, we have investigated the timing of DNA duplication in the mouse zygote. While we were analysing our data, Luthardt & Donahue (1973) published an extensive work on pronuclear DNA synthesis in the mouse. Their findings will be discussed.Virgin Swiss female mice (CD-I COBS) purchased from Charles River Italia were kept in a room with controlled temperature

We compared the frequency of malignancies in 207 multiple sclerosis patients (mean age 35.75 years, SD 10.60) who took 2.0 mg/kg azathioprine daily (mean duration 4.16 years; SD 2.38) and in 247 nontreat-ed patients (mean age 35.44 years; SD 11.94). Five malignancies were diagnosed in the azathioprine group compared with seven in the control group. The age-adjusted occurrence rate was 3.62/1,000 person-years (95 CI, 1.17 to 8.43) in the treated and 4.24/1,000 person-years (95 CI, 1.70 to 8.73) in the nontreated group; the age-adjusted relative risk of developing a tumor was 0.85.

Institutes of Histology and General Embryology of the University of Rome, and of the School of Medicine of L'Aquila, Italy(Received 29th November 1974)In a previous paper (Siracusa, 1973) we have shown that the variations in the level of total RNA polymerase activity per embryo and per blastomere in the preimplantation mouse embryo are not correlated with patterns of RNA synthesis at corresponding stages of development. We concluded that, in the mouse embryo, in contrast with the situation found in the sea urchin embryo (Roeder & Rutter, 1970), RNA polymerase apparently does not play any direct regulatory rôle in RNA synthesis.The possibility remained, however, that a correlation could still exist for only one of the two main forms of the enzyme. A situation of this kind has been found during the development of Rana pipiensembryos (Kohl, Norman & Brooks, 1973); Polymerase I (the nucleolar form) and Polymerase

Institutes of Histology and General Embryology, University of Rome, and School of Medicine of L'Aquila, ItalyIshida & Chang (1965) found that hamster and rabbit embryos show a gradual increase of the activity of the mitochondrial enzyme succinate dehydrogenase (SDH: E.C. 1.3.99.1) between the 1- and 8-cell stages, followed by a steeper rise at blastulation. Baranska, Dorywalski & Kujawa (1973) reported that SDH activity increases from the 1- to 4-celled stage and decreases in the morula and blastocyst in the mouse. In both these studies SDH was demonstrated by the Nachlas method (Nachlas, Tsou, Souza, Cheng & Seligman, 1957), a histochemical reaction based on the reduction of nitro blue tetrazolium (NBT), in which no intermediate electron acceptor between SDH and the tetrazolium dye is used. However, Horwitz, Benitez & Bray (1967) have shown that SDH cannot transfer electrons directly to NBT, but only by way of coenzyme Q or another