Audit of newborn screening programme for congenital hypothyroidism.

Newborn screening for congenital hypothyroidism (CH) was implemented in Hospital UKM in December 2004 using cord blood sample. From the audit over a period of 25 months, a total of 13,875 newborn babies were screened with a coverage of 98.8%. From this cohort, the mean recall rate was 0.32%; unfortunately the mean percentage of recalled babies that came for retesting was only 79.5%. In addition, the mean sample rejection rate was high, i.e. 2.2%. Two babies were diagnosed to have CH. These findings implied that whilst the coverage of screening was good, there is a need for regular surveillance of performance of both clinical and laboratory personnel. In addition, a more concerted effort should be carried out to promote community awareness of such a programme.

A rare case of ambiguous genitalia.

Genes on the Y chromosome are essential for normal sex determination and sex differentiation of male genitalia. However, genes on the X chromosome and other autosomes have been shown to be anti-testes and have a detrimental effect on this process. Addition of X chromosomes to the 46,XY karyotype results in seminiferous tubules dysgenesis, hypogonadism and malformed genitalia. We report a term male newborn with 49,XXXXY syndrome presenting with ambiguous genitalia, multiple extra-gonadal anomalies, facial dysmorphism, and radioulnar synostosis.

A case-control study of risk factors associated with rectal colonization of extended-spectrum beta-lactamase producing Klebsiella sp. in newborn infants.

To determine the risk factors for rectal colonization by extended-spectrum beta-lactamase (ESBL) Klebsiella sp. in 368 newborns admitted consecutively to a neonatal intensive care unit over six months, rectal swabs were cultured on admission and weekly until discharge. Eighty infants (21.7%) had ESBL Klebsiella sp. cultured from their rectal swabs. Eighty controls were selected at random from infants with negative cultures admitted within the 14-day period prior to the detection of ESBL Klebsiella sp. in the cases. Cases had significantly lower birth weight, gestational age, earlier age of admission, longer hospital stay, and higher proportions of congenital malformations, early-onset pneumonia and respiratory distress syndrome compared with controls. Significantly more cases received mechanical ventilation, nasal continuous positive airway pressure support, total parenteral nutrition, umbilical vascular catheterization, arterial line insertion, urinary bladder catheterization, and prior treatment with antibiotics. However, stepwise logistic regression analysis showed that only two independent risk factors were significantly associated with ESBL rectal colonization: duration of hospital stay [adjusted odds ratio (OR): 1.3; 95% confidence intervals (CI): 1.2, 1.4; P<0.0001) and early-onset pneumonia (adjusted OR: 8.3; 95% CI: 1.6, 43.4; P=0.01).

Identifying HIV/AIDS primary care development needs.

AIM: This paper reports a study aimed at identifying the primary health care experiences of people living with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) in Malaysia. The rationale behind the study was to enable informed action for developing more responsive and effective primary care. BACKGROUND: Reports such as from the World Health Organisation forecast sharp escalations in the incidence of HIV/AIDS in Malaysia and the Asia-Pacific region within the next few years. With sparse information on the course of infection on the local population and an understanding of health care needs of those afflicted, health services would be ill-prepared for projected increases. METHOD: Semi-structured interviews were conducted with a convenience sample of 99 patients attending two major HIV/AIDS clinics in Malaysia. FINDINGS: Several gaps in care provision were highlighted, such as with treatment/consultation facilities and availability and accessibility of information. What is also evident is that there are a number of good support services available but not well publicized to those in need of them. That includes health professionals who could be making appropriate referrals. The lack of communications and inter-professional working appears to be part of the problem. CONCLUSION: The findings provide baseline data and preliminary insights to government and other service providers towards advancing, optimizing and refining existing policies and infrastructure. Although the availability of a number of primary care facilities have been identified, the study indicates the need for more effective co-ordinated efforts with clear leadership to pull together scarce resources towards the aim of some degree of seamless primary care provision. It is suggested that nurses would be well placed for such a role in view of the nature of their education and training that helps prepare them for the multi-faceted role.

Infantile isolated sulphite oxidase deficiency in a Chinese family: a rare neurodegenerative disorder.

We report the clinical, biochemical, neuroradiological, and neurophysiological findings of a 4-year-old Chinese girl with infantile isolated sulphite oxidase deficiency. This is the first reported case in our locality. She presented at the age of 5 months with refractory seizures and developmental regression, and progressed rapidly to profound psychomotor retardation, spasticity, dystonia, microcephaly, and blindness. At the age of 3.5 years, she was admitted to the intensive care unit with septic shock. Ophthalmologic examination at this time revealed bilateral dislocation of the lens. Diagnosis of this very rare disorder was made on the basis of increased levels of urinary sulphite, thiosulphate, and sulphocysteine; normal urine xanthine and hypoxanthine; normal plasma uric acid; and low plasma cystine levels. The diagnosis was confirmed by the absence of sulphite oxidase activities in skin fibroblasts. Isolated sulphite oxidase deficiency is a rare inborn error of sulphur metabolism that is difficult to diagnose on clinical features and routine metabolic tests. The presence of ectopia lentis, seizures, and progressive neurological abnormalities should alert clinicians to the diagnosis.

Hormonal perturbations in patients with testicular cancer treated with cisplatin.

Patients with testicular cancer treated with cisplatin can undergo feminization that is understood poorly. Rat model studies recently showed that cisplatin can feminize in part the profile of hepatic steroid-metabolizing enzymes and circulating hormone levels. This study was undertaken to determine whether cisplatin similarly might contribute to the perturbations in gonadotropin or steroid hormone levels that can occur in patients undergoing cisplatin-based treatment for testicular cancer. Analysis of serum free testosterone, total testosterone, and androstenedione levels revealed that these hormones were not altered significantly in patients during a 38-week period of cisplatin-based treatment and follow-up. Estradiol levels were elevated before chemotherapy and were reduced to normal levels during treatment. This reduction was attributed to the cytotoxic effect of chemotherapy on the tumors and the resultant reduction in serum chorionic gonadotropin levels. Serum dihydrotestosterone (DHT) levels were normal before chemotherapy but progressively became elevated during treatment with cisplatin in five of ten patients examined. The rise in DHT may relate to the previously described increase in hepatic androgen 5 alpha-reductase activity in cisplatin-treated rats. Levels of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone (FSH) were normal before cisplatin-based treatment was administered; however, FSH was elevated selectively during chemotherapy. This selective induction of FSH may reflect an effect of cisplatin on the hypothalamic secretion of gonadotropin-releasing hormone. Taken together, these findings suggest that cisplatin contributes to the perturbation of steroid and peptide hormone levels in patients with testicular cancer and perhaps in others undergoing cisplatin-based chemotherapy.

Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes.

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

N,N',N''-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents.

The cancer chemotherapeutic drug N,N',N''-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N''-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate.

Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketoglutarate (domain I); 2) the C1-C2 keto acid moiety of alpha-ketoglutarate (domain II); 3) the distance between domains I and II; and 4) the spatial relationship of the two domains required for optimal interaction with the cosubstrate binding site. All compounds were competitive inhibitors for alpha-ketoglutarate (Km 0.018 mM). Functionally, two subsites of the cosubstrate binding site were evident: subsite I for polar interaction with the C5 carboxyl group, and subsite II, comprising of two distinct cis-oriented coordination sites of the catalytic ferrous ion which interact with the C1-C2 keto acid moiety. The most efficient inhibitors were pyridine 2,4-dicarboxylate (Ki 0.0002 mM) and 3,4-dihydroxybenzoate (Ki 0.0006 mM). Both compounds contain a carboxyl group and a chelating moiety corresponding to domains I and II of alpha-ketoglutarate, respectively. The fixed orientation of these groups in both analogs was used to assess intersubsite distance and spatial relationship required for optimal interaction with the cosubstrate binding site. Binding at subsite I and chelation at subsite II were indispensible for effective competitive inhibition. The distance between these two domains also helped determine whether attachment at the cosubstrate binding site would be catalytically productive. This was emphasized by the failure of either oxaloacetate or alpha-ketoadipinate to promote hydroxylation. Optimal interdomain distance, however, was not sufficient for cosubstrate utilization, as pyridine 2,4-dicarboxylate, with an interdomain distance identical to alpha-ketoglutarate in its staggered conformation, did not sustain hydroxylation. In the overall, these studies suggest that alpha-ketoglutarate utilization occurs in a ligand reaction at the active site ferrous ion of gamma-butyrobetaine hydroxylase. This is of particular interest since the delineated stereochemical mode of oxidative decarboxylation could generate the reactive oxo-iron species that was shown experimentally to promote gamma-butyrobetaine hydroxylation by an abstraction-recombination mechanism (Blanchard, J. S., and Englard, S. (1983) Biochemistry 22, 5922-5928; Englard, S., Blanchard, J. S., and Midelfort, C. F. (1985) Biochemistry 24, 1110-1116).

Somatic function of the micronucleus of Stylonychia mytilus during asexual propagation.

The somatic function of the micronucleus during vegetative propagation of the hypotrichous ciliate Stylonychia mytilus was investigated, by generating amicronucleate cell lines with microinjection or amputation. The amicronucleate cell lines exhibited reduction in fission rate and ingestion shortly after the loss of the micronucleus. There was partial recovery of growth rate in the following months of propagation. Amicronucleate cultures carried both normal-looking and abnormal cells. The normal-looking ones underwent binary fission, and also physiological reorganization, in the usual manner. After binary fission, normal-looking amicronucleates gave rise to normal-looking post-dividers, which might then undergo abnormal transformation in the interfission period by developing a bulge on the posterior-right side of the cell (Type A), or on the dorsal surface (Type B). Type A cells could enter binary fission directly, eventually recovering normal cell shape. The hump-back Type B cells went through atypical cortical reorganization(s) to generate spherical cells, which then recover to normal shape by normalizing reorganization(s). The emergence of these two types of abnormalities was correlated with the failure of macronuclear division during binary fission. These observations indicate that the micronucleus possesses morphogenetic function, and that this function is division-related. The micronucleus probably affects cytoskeletal/membranellar development during binary fission, and in its absence the cortical abnormalities exhibited represent a delayed expression of the infirmities incurred during binary fission. In view of the persistence of such abnormalities in amicronucleate cultures for up to a year of culture, the morphogenetic function of the micronucleus appeared not to be replaced; this resembles the situation in the hypotrich Pseudourostyla cristata, but differs from Paramecium where recovery to near-normal is the rule. Chromatin bodies resembling pseudomicronuclei were present in low frequency in the culture, and these appeared to arise in connection with macronuclear development in Type B cells.

Discrimination of cytoskeletal elements of Paramecium by heterologous antisera: A preliminary investigation on the presence of intermediate filament-related protein.

The present study of the cytoskeleton of Paramecium employed a number of heterologous monoclonal antisera raised against intermediate filament proteins of higher organisms, and also an antiserum against a 49 K protein from Tetrahymena. Anti-49 K, and also monoclonal anti-bovine cytokeratin K 8.13 which has a broad cross-reaction spectrum for epithelial intermediate filaments, differentiated the microtubular systems of Paramecium into two categories. Those closely associated with surface membranes, such as basal bodies, contractile vacuole pores and the cytospindle, and the gullet also, were labeled by these antibodies. Others internal to the epiplasm are not labeled, such as transverse and postciliary microtubular bands associating with basal bodies, contractile vacuole canals, cytopharyngeal ribbons and postoral fibers. None of the heterologous anti-tubulins tested in this and previous studies generated such a pattern of discrimination of the microtubular systems of Paramecium, indicating that the discrimination by anti-49 K and by anti-cytokeratin K 8.13 was not based on the molecular composition (α- or ß-tubulin) of the microtubular systems, their state of tyrosination, or acetylation. These two antibodies thus reveal a determinant that is associated exclusively with certain membrane-bound microtubular systems of Paramecium. This determinant appears to participate in the development of nascent structures like the cytospindle and the oral apparatus, since it co-distributed with these structures in their different stages of development. In addition, two monoclonal antibodies were superimposable specifically on certain cytoskeletal elements also residing close to surface membranes: anti-pig vimentin V 9 labeled the epiplasm underlying the inner alveolar membrane, while anti-human cytokeratin K 4.62 labeled the outer lattice lining the cortical ridges. On the other hand, anti-49 K labeled the micronucleus and its derivatives in all stages of sexual nuclear reorganization in Paramecium, unlike the specific labeling of postmeiotic and zygotic nuclei in Tetrahymena. The presence of intermediate filament-related protein in Paramecium is discussed.

Nuclear control of cortical reorganization during sexual reproduction of Stylonychia mytilus: A study with amicronucleates.

During conjugation, Stylonychia mytilus undergoes repetitive somatic development by reorganizing the cortical ciliature consecutively for three times. The control of these reorganizations by the nucleus was investigated employing amicronucleates generated by microsurgery. When amicronucleates conjugate, they underwent only the first round of cortical reorganization; the exconjugants perished before the second cortical reorganization commenced. This shows that the micronucleus is dispensable for the first cortical reorganization, but that the micronucleus, or its divisional derivatives in sexual nucleogenesis, are essential for immediate postconjugational survival. Conjugation of amicronucleates with micronucleates in most cases resulted in the arrest of macronuclear anlage development at the post-polyteny DNA-poor stage; the second cortical reorganization could not be initiated in such cells. In others where the macronuclear anlage developed beyond the post-polyteny DNA-poor stage, the cell underwent the second, and then the third round of cortical reorganization. This correlation between macronuclear anlage development and the second cortical reorganization argues for a causal relationship between the two. Very likely, the macronuclear anlage up to the post-polyteny DNA-poor stage provides the crucial morphogenetic signal(s) for the second cortical reorganization. The nature of the first and second rounds of cortical reorganization are thus fundamentally different from each other, as their nuclear controls are radically dissimilar.

Effect of biosynthetic human growth hormone on insulin action in individual tissues of the rat in vivo.

Excessive endogenous production or exogenous administration of human growth hormone (hGH) causes insulin resistance at both the hepatic and extrahepatic levels. However, which extrahepatic tissues are involved have not been defined. We have examined the diabetogenic action of authentic biosynthetic hGH on whole body glucose disposal, hepatic glucose output, and glucose metabolism in individual peripheral tissues. The use of a highly purified preparation of the hormone allowed us to examine the isolated effects of 22K hGH. The euglycemic hyperinsulinemic (approximately 100 mU/L) clamp plus 3H-2-deoxyglucose technique was used to quantitate the effects of hGH on insulin action in vivo. Administration of biosynthetic hGH at a dose of 10 IU/kg/24 h for 48 hours in male Wistar rats (approximately 340 g) produced a highly significant decrease in the steady state clamp glucose infusion rate (GIR) when compared with controls (8.1 +/- 0.6 v 18.7 +/- 0.7 mg/kg/min, P less than .001), reduced insulin-mediated suppression of hepatic glucose output (Ra) (3.9 +/- 0.6 v 0.7 +/- 0.3 mg/kg/min, P less than .05) and a decreased clamp glucose disposal rate (Rd) (12.0 +/- 0.4 v 18.10 +/- 1.1 mg/kg/min, P less than .001). There was a significant decrease in insulin-mediated glucose uptake as indicated by tissue accumulation of [3H]-2-deoxyglucose phosphorylation in diaphragm and hindlimb muscles. Insulin action was more substantially reduced in muscles (approximately 50%) than in adipose tissues (approximately 20%). These studies confirm that the diabetogenic action of hGH in the rat is due to a combination of inhibition of insulin suppression of hepatic glucose output and inhibition of the uptake and subsequent utilization of glucose in skeletal muscles.

Biotransformation of N,N',N''-triethylenethiophosphoramide: oxidative desulfuration to yield N,N',N''-triethylenephosphoramide associated with suicide inactivation of a phenobarbital-inducible hepatic P-450 monooxygenase.

Oxidative metabolism of the polyfunctional alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N''-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (Km (app) 19 microM; Vmax (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by kinactivation = 0.096 min-1, KI = 24 microM, and a partition ratio of 136 +/- 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-hydroxysteroid:NADP+ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification of the surface membrane-cytoskeleton complex (Cortex) of Paramecium and identification of several of its protein constituents.

In ciliates, the major morphogenetic events take place in the cortex, a complex of membranes and closely associated filamentous networks. To analyze the problems of assembly and morphogenesis at the molecular level in Paramecium, we have developed a method of purification of cortex fragments which retain their in situ organization and display a highly reproducible electrophoretic profile. The method used either a four-step sucrose gradient yielding a cortex + oral apparatus fraction or a six-step gradient which allowed the cortex fragments to be freed from the oral apparatuses (which were recovered separately). By comparative electrophoresis and immunological probing of these and other cell fractions or purified organelles, we could identify several of the major polypeptides resolved by SDS PAGE as components of specific cortical or oral structures. The purification method was successfully applied to morphological mutants, and the first case of a mutational modification of a cortical polypeptide was observed.

Programming of the macronucleus of Paramecium during asexual and sexual reproduction: A further study with cytidine analogues, dimethylsulfoxide, L-ethionine and N-butyric acid.

The control of the function of the macronucleus of Paramecium is studied, in connection with its role in the compensation for the asexual somatic function of the micronucleus. Following removal of the micronuclei, amicronucleate cell lines as a rule suffer a transient period of growth and developmental depression in the initial phase of asexual propagation. But they gradually recover to near-normal. Previous studies of treatment of amicronucleate cells with cytidine analogues have implicated the macronucleus in compensating for the somatic function of the micronucleus following the loss of the micronucleus, and the activation of this macronuclear function probably involves DNA-demethylation. The present study further tests this notion, by treating micronucleate cells with agents known to promote demethylation of 5-methylcytosine. After treatment, the cells were vegetatively propagated, and then enucleated to give rise to amicronucleate cell lines. Treatments with dimethylsulfoxide, L-ethionine, and 5-aza-2'-deoxycytidine promoted recovery in amicronucleate cell lines thus derived. Cells treated with 6-azacytidine did not produce such an effect. Hence, the compensatory mechanism, presumably residing in a repressed state in the macronucleus, can be activated or primed to activate by demethylating agents even before the loss of the micronucleus, and once established the new macronuclear programme perpetuates in succeeding asexual cell generations. This shows that during asexual propagation the macronuclear programme can be altered to 'pre-adapt' the cells for amicronuclearity. Treatment of micronucleate conjugants with 5-azacytidine, when the macronuclear anlagen develop, produced clones that had become similarly pre-adapted. There were also some indication of persistence of such effects of the analogue into the next clonal cycle following autogamy. The notion of macronuclear DNA-demethylation as a basis for the activation and maintenance of the compensatory mechanism is discussed.

Repressive function of the micronucleus during asexual reproduction in Paramecium tetraurelia: Experimental analysis with defective-micronucleates generated by laser microbeam irradiation.

The present paper reports on the experimental analysis of a novel regulatory function of the micronucleus of Paramecium tetraurelia. Previous studies have made clear that amicronucleate cell lines shortly after their generation generally suffer depression, in exhibiting low viability, slow growth and abnormal oral development in binary fission, but they eventually recover to near-normal. A compensatory mechanism is thus activated in the absence of the micronucleus, to allow recovery of the amicronucleate cell line. Implicit in this conclusion is the role of the micronucleus in repression of the compensatory mechanism. The present study tested this notion by perturbing the micronucleus with laser microbeam irradiation. This operation generated cell lines possessing defective micronuclei; during their asexual propagation, some cells lost the micronucleus and gave rise to amicronucleates. The viability of amicronucleate cell lines derived in this manner was found to be higher, compared to others generated by a different operation involving instantaneous removal of normal micronuclei from the cell with a microinjection needle. Some evidence also suggested that their oral development was less abnormal during the initial depression period. Hence, damage of the micronucleus has apparently facilitated the activation of the compensatory function, and the latter might have occurred even before the loss of the defective micronucleus. The present findings provide support for a regulatory role of the micronucleus during asexual propagation. Previous studies have indicated that the physical basis of the compensatory mechanism resides with the macronucleus. The micronuclear repressive function may be directed against this compensatory mechanism of the macronucleus.

Control of development of the oral apparatus of Paramecium during sexual reproduction: an embryological perspective.

This study shows that development of the new soma during sexual reproduction in ciliates can be conceptualized on the same basis as embryogenesis in multicellular organisms. In conjugating Paramecium, development of a new oral apparatus takes place during fertilization and the first three divisions of the zygotic nucleus and completes well before the postsexual cell undergoes the first cell fission. The control of oral development is analyzed by microsurgical removal of the zygotic nucleus or the postzygotic nuclei from conjugants. The enucleated exconjugants can pass through an early hurdle in oral development (the initiation of oral membranelle assembly) and subsequently develop an oral apparatus. Such oral apparatuses nevertheless exhibit structural and functional abnormalities including fragmentation and misalignment of oral membranelles, absence of the postoral microtubular bundle, reduction in the length of buccal cavity, and impaired phagocytosis. Other stomatogenic aspects, such as the arrangement of basal bodies in the oral membranelles, remain unaffected. The two groups of exconjugants, one derived from cells enucleated at the zygotic stage, and the other at the postzygotic stage, exhibit the same types of oral abnormality. We conclude that (i) the zygotic nucleus is not essential for the initiation of oral membranelle assembly. The existence of zygotic signals for subsequent oral development is not ruled out, but these are insufficient. (ii) Postzygotic nuclei, as well as maternal nuclei (the old somatic nucleus and meiotic derivatives of the germ nucleus), control oral development. This reveals a parallelism between postsexual development in ciliates and the early embryology of multicellular organisms, in their reliance on information provided by maternal, as well as early postzygotic nuclei. (iii) The activity of the old somatic nucleus alone is not sufficient for the later stages of oral development. Probably, some stomatogenic functions of the old somatic nucleus normally utilized for the later stages of oral development in binary fission are inactivated during sexual reproduction. Alternatively, the old somatic nucleus may rely on some critical conditions prescribed by the postzygotic nuclei in order to act.

5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium.

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.