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L C Smith

Publications and source records attributed to L C Smith.

At least 19 recordsLinked to original sources
PubMed1992

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Mouse macrophage receptor for acetylated low density lipoprotein: demonstration of a fully functional subunit in the membrane and with purified receptor.

The functional molecular mass of the macrophage receptor for acetylated low density lipoprotein (Ac-LDL) was determined in membranes by radiation inactivation analysis. Membranes from tumors induced by the mouse macrophage cell line P388D1 were frozen and irradiated with high-energy electrons. Residual binding activity indicated a minimum functional molecular mass of 35,000 Da, considerably smaller than the active 260,000 M(r) protein seen on ligand blots under nonreducing conditions. Scatchard analysis of receptor binding gave no evidence of partially inactivated molecules. The receptor protein, purified by affinity chromatography and preparative gel electrophoresis, was incubated with dithiothreitol (0.1-100 mM) and retested for binding activity. Active subunits of 158,000 and 80,000 M(r) could be demonstrated by ligand blotting, with quantitative conversion of binding activity to the 80,000 M(r) species at 10 mM dithiothreitol. At 100 mM dithiothreitol, all binding activity was lost. Further size reduction was not detected by silver staining. These data suggest that the isolated mouse macrophage Ac-LDL receptor is a trimer with one class of SH groups involved in trimerization and another in the actual binding site. The monomeric species is fully active in vitro under mild reducing conditions. The radiation inactivation data also suggest that each monomeric unit is fully active and capable of functioning independently in the binding of ligands in the membrane.

Animals↗
PubMed1992

Demography of the Hadza, an increasing and high density population of Savanna foragers.

This is a report on the demography of the Hadza, a population of East African hunter-gatherers. In it, we describe the results of a census, and our estimation of age structure, survivorship, mean age of women at childbearing, number of live children, total population size and density, and rate of change since 1967. We show that relevant measures fit closely the stable population model North 6 chosen by Dyson to represent Hadza demography in the 1960s. We compare aspects of Hadza demography with surrounding non-Hadza and with the !Kung. Among other things, we find that the Hadza have a higher population density, higher fertility, and a faster population growth rate than do the !Kung. These demographic differences are consistent with our expectations, which were based on differences in the costs and benefits of foraging in the two regions. We also show that Hadza demographic parameters display remarkable consistency over the past 20 years. Since neighboring populations have been encroaching on the area used by the Hadza, and Hadza foragers have been subject to interludes of externally imposed settlement, this consistency is surprising. We discuss some of the implications.

Adolescent↗
PubMed1992

SpCoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury.

SpCoel1 is a single copy gene that is specifically expressed in most of the coelomocytes of the adult purple sea urchin, Strongylocentrotus purpuratus. The 4-kb transcript from this gene has a relatively short (426 nucleotide) open reading frame (ORF) with long 3' and 5' untranslated regions. The ORF encodes a protein that has strong amino acid sequence similarity to profilins from yeast to mammals. Transcript titrations of SpCoel1 show significant increases per coelomocyte in animals that have been physiologically challenged. Increases in transcript levels are of similar magnitudes between animals receiving different treatments, such as injuries from needle punctures or from injections of foreign cells. The evidence presented here implies a molecular mechanism by which this lower deuterostome defense system responds to external insult, viz that an external "injury signal" activates a signal transduction system, which in turn mediates the alterations in cytoskeletal state that are required for coelomocyte activation.

Amino Acid Sequence↗
PubMed1992

Ontogeny, immunocytochemical localization, and biochemical properties of the pregnancy-associated uterine elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP): monospecific antibodies to a synthetic peptide recognize native ALP.

Expression of the mRNA encoding the elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP), is highest in pig uterus during mid- and late pregnancy, suggesting a stage of pregnancy-dependent role for ALP in feto-maternal interactions. To elucidate a function for ALP in these events, immunogenic probes were developed to localize sites of ALP expression in the environment of the developing fetus. Monospecific antibodies raised against a 16-mer synthetic peptide corresponding to residues 21-36 (ALP 16P) of the deduced amino acid sequence of pig uterine ALP were generated by active immunization of sheep. ALP 16P conjugated to keyhole limpet hemocyanin elicited high titer antibodies that were specific to ALP. The antipeptide antibodies were used to characterize pig uterine ALP from allantoic fluids. Uterine ALP has an approximate mol wt of 14,000 and a pI of 8.2 and exhibits elastase inhibitor activity. Amino-terminal amino acid sequencing of uterine ALP indicated the sequence AENALKGGACPPRKIVQC, which has 44% identity with the corresponding region in human bronchial ALP. RIA for ALP, developed using ALP 16P as standard and iodinated tracer, demonstrated the presence of immunoreactive ALP in early, mid-, and late pregnant endometrium and myometrium, placenta, allantoic fluids, fetal cord blood, and fetal liver. ALP was undetectable in the maternal circulation. The ALP levels in endometrium, allantoic fluids, and fetal cord blood changed with the stage of pregnancy; however, ALP content in placenta, myometrium, and fetal liver, although different among tissues, remained invariant during gestation. By immunocytochemical analyses, ALP was localized in the glandular epithelium of the uterus, in placenta, and in fetal liver, consistent with the presence of immunoreactive ALP as measured by RIA. The localization of uterine ALP in placenta and its corresponding transport to fetal circulation provide strong evidence to support a physiological function for the protease inhibitor in the biological mechanisms controlling fetal development in utero.

Amino Acid Sequence↗
PubMed1992

A technical-skills course for 1st-year residents in general surgery: a descriptive study.

The proper teaching of operative skills to surgical residents is increasingly constrained by operative time, complex procedures and medicolegal concerns. A technical-skills program was developed and introduced over a 3-year period to 28 1st-year residents in general surgery. To introduce the residents to the principles of surgical techniques in a simulated environment outside the operating room, the program consisted of a combination of two didactic sessions and six "wet labs" taking 3 to 4 hours per week for 8 weeks between January and March each year. The didactic sessions included instruction on suture material and the use of stapling devices; the "wet lab" used a "hands-on" approach. Educational objectives in the "wet lab" included instruction on preparation of the patient and draping, aseptic technique, principles of bowel anastomosis, incisions, instrument use and handling, principles of hemostasis, intraoperative surgical emergencies, surgical assisting and overall conduct in the operating room. The residents' surgical technique and skills improved over the course period. The overall value, teaching and understanding of surgical principles were rated highly. Problems cited during resident feedback were the use of live animals and insufficient time to practise. The efficacy of a surgical-skills program has been demonstrated, but its effectiveness requires further evaluation.

Canada↗
PubMed1992

Different reactivities of high density lipoprotein2 subfractions with hepatic lipase.

Human high density lipoproteins2 (HDL2) consist of particles that contain both apolipoprotein (apo) A-I and apoA-II (A-I/A-II-HDL2) and others that contain apoA-I but are devoid of apoA-II (A-I-HDL2). When postprandial lipemia is pronounced, a fraction of HDL2 is converted into HDL2-like particles. These HDL3 exhibit lower apoA-I/apoA-II ratios than the parent HDL2, suggesting preferential conversion of A-I/A-II-HDL2 into HDL3 (J. Clin. Invest. 1984. 74: 2017-2023). Triglyceride transfer from triglyceride-rich lipoproteins to HDL2 and subsequent lipolysis by hepatic lipase are thought to mediate the conversion of HDL2 into HDL3. To understand why A-I/A-II-HDL2 are preferentially converted into HDL3, we separated postprandial HDL2 into A-I-HDL2 and A-I/A-II-HDL2 species by immunoaffinity chromatography using a monoclonal antibody for apoA-II, and determined the ability of HDL2 species i) to participate in protein-mediated lipid transfer; and ii) to interact with hepatic lipase in vitro. Triglyceride transfer from/to triglyceride-rich lipoproteins was similar for the two HDL2 species. In contrast, A-I/A-II-HDL2 were twice as effective as A-I-HDL2 in liberating hepatic lipase immobilized on HDL3-Sepharose. Lipolysis of triglycerides by hepatic lipase was 60% higher in postprandial A-I/A-II-HDL2 than in postprandial A-I-HDL2. Hydrolysis of phosphatidylcholine by hepatic lipase was threefold higher in A-II-containing HDL2 when compared with HDL2 devoid of apoA-II. The different lipolytic rates in HDL2 subspecies correlated with the size reduction of substrate lipoproteins. Reconstitution of postprandial A-I-HDL2 with apoA-II enhanced the rate of lipolysis by hepatic lipase to that observed in A-I/A-II-HDL2. We conclude that it is the interaction with hepatic lipase rather than the rate of triglyceride transfer that results in the preferred conversion of postprandial A-II-containing HDL2 into HDL3, and that apoA-II exerts a crucial role in this process.

Apolipoprotein A-I↗
PubMed1992

A missense mutation (Trp86----Arg) in exon 3 of the lipoprotein lipase gene: a cause of familial chylomicronemia.

We have investigated a patient of English ancestry with familial chylomicronemia caused by lipoprotein lipase (LPL) deficiency. DNA sequence analysis of all exons and intron-exon boundaries of the LPL gene identified two single-base mutations, a T----C transition for codon 86 (TGG) at nucleotide 511, resulting in a Trp86----Arg substitution, and a C----T transition at nucleotide 571, involving the codon CAG encoding Gln106 and producing Gln106----Stop, a mutation described by Emi et al. The functional significance of the two mutations was confirmed by in vitro expression and enzyme activity assays of the mutant LPL. Linkage analysis established that the patient is a compound heterozygote for the two mutations. The Trp86----Arg mutation in exon 3 is the first natural mutation identified outside exons 4-6, which encompass the catalytic triad residues.

Amino Acid Sequence↗
PubMed1992

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.

Amino Acid Sequence↗
PubMed1991

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family.

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1991

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis.

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.

Amino Acid Sequence↗
PubMed1991

Nutritional assessment and indications for nutritional support.

The goal of nutritional assessment is to identify prospectively all those patients who would develop a nutrition-related complication. In practical terms, there is no single test capable of achieving this goal. At present, the best method of nutritional assessment is an organized step-by-step multifactorial approach. This involves assessment of the primary illness, the patient history, and the prognosis. A nutritional status examination is done, and the current intake is compared with the nutritional goals. A decision is then made whether to force feed. If forced feeding is initiated, the effectiveness of the therapy must be monitored frequently until the patient recovers and is able to be sustained by volitional oral intake.

Enteral Nutrition↗
PubMed1991

Human immunodeficiency virus (HIV) transcripts identified in HIV-related psoriasis and Kaposi's sarcoma lesions.

Persons with HIV infection sometimes develop aggressive psoriasis or Kaposi's sarcoma (KS) not usually seen in other immunosuppressed patients. However, a specific and direct pathophysiological role for HIV-1 in these AIDS-associated disorders remains unclear since HIV has not been easily detected in these skin lesions. By combining in situ hybridization with the sensitive detection technique of confocal laser scanning microscopy, we have demonstrated HIV RNA transcripts in 5 of 15 lesional skin biopsies from HIV-infected psoriasis patients, and in 3 of 8 Kaposi's sarcoma biopsies from HIV-infected patients. HIV transcripts were not detected in normal appearing skin from HIV-infected patients or in psoriatic and normal skin biopsies from uninfected individuals (P = 0.006). Although previous attempts to demonstrate viral sequences in psoriasis and KS lesions have been unsuccessful, in situ hybridization with confocal microscopy has shown the presence of HIV RNA transcripts predominantly within CD4+, Factor XIIIa positive dermal dendrocytes. HIV or cytokines produced by infected cells in skin lesions may therefore play a direct role in the pathogenesis of HIV-associated psoriasis and KS.

Adult↗
PubMed1990

In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.

Amino Acid Sequence↗
PubMed1990

Confocal microscopic detection of human immunodeficiency virus RNA-producing cells.

A central anomaly in the pathogenesis of AIDS is that few actively infected CD4+ cells (1 in 10(4)-10(5) have been detected in the peripheral blood, even though dramatic depletion (often greater than 90%) of CD4+ cells is the hallmark of disease progression. A sensitive, 35S-based human immunodeficiency virus (HIV) RNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. In 35 symptomatic HIV-infected individuals (AIDS and AIDS related complex), an average of 1 in 350 mononuclear cells produced HIV RNA. In contrast, in an asymptomatic group of 30 individuals, an average of 1 in 2000 mononuclear cells produced HIV RNA. These data, obtained using this improved detection method, suggest there are more HIV RNA-producing cells in HIV-infected individuals than previously reported. In addition, increased numbers of HIV transcribing cells were found to correlate with declining clinical condition as assessed by Karnofsky performance score. These data suggest that viremia per se may account for the pathologic consequences in HIV infection.

AIDS-Related Complex↗
PubMed1990

Retinol-binding protein: a major secretory product of the pig conceptus.

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.

Amino Acid Sequence↗
PubMed1990

Control of first cleavage in single-cell reconstituted mouse embryos.

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.

Animals↗