Intrauterine devices: effects on ultrastructure of human endometrium.

The human endometrium, studied with the electron microscope, undergoes asynchronous and premature cyclic development in response to inin uterine contraceptive devices. Typical nucleolar channel systems and other cellular characteristics of the normal secretory phase appear before ovulation, and decidualization occurs several days prematurely. Disturbance of the synchrony of ovular and endometrial development may be a mechanism of contraceptive action of these devices.

Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.

Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease.

BACKGROUND: Mutations in components of the extraordinarily large alpha-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain alpha-ketoacid dehydrogenase (E1), the 170 kDa alpha(2)beta(2) heterotetrameric E1b component of the branched-chain alpha-ketoacid dehydrogenase multienzyme complex. RESULTS: The 2.7 A resolution crystal structure of human E1b revealed essentially the full alpha and beta polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K(+)) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the beta subunit near the interface with the small C-terminal domain of the alpha subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K(+) sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr-->Asn mutation at position 393-alpha occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b alpha subunit, contacting the beta' subunit. CONCLUSIONS: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD.

Differential processing of human and rat E1 alpha precursors of the branched-chain alpha-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence.

The N-terminal sequences of the E1 alpha, E1 beta and E2 subunits of the human branched-chain alpha-keto acid dehydrogenase complex have been determined by microsequencing. The N-terminal of human E1 beta and E2 subunits (Val and Gly, respectively) are identical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 alpha subunit (Ser) is identical to bovine, but differs from the rat E1 alpha (Phe) subunit. Comparison of the N-terminal sequences of human and rat E1 alpha subunits shows that the serine residue at the +1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5'-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-Pro bond, similar to trypsin, chymotrypsin and microsomal signal peptidases.

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

The Rhodospirillum rubrum cytochrome bc1 complex: peptide composition, prosthetic group content and quinone binding.

A cytochrome bc1 complex, essentially free of bacteriochlorophyll, has been purified from the photosynthetic purple non-sulfur bacterium Rhodospirillum rubrum. The complex catalyzes electron flow from quinol to cytochrome c (turnover number = 75 s-1) that is inhibited by low concentrations of antimycin A and myxothiazol. The complex contains only three peptide subunits: cytochrome b (Mr = 35,000); cytochrome c1 (Mr = 31,000) and the Rieske iron-sulfur protein (Mr = 22,400). Em values (pH 7.4) were measured for cytochrome c1 (+320 mV) and the two hemes of cytochrome b (-33 and -90 mV). Electron flow from quinol to cytochrome c is inhibited when the complex is pre-illuminated in the presence of a ubiquinone photoaffinity analog (azido-Q). During illumination, the azido-Q becomes covalently attached to the cytochrome b peptide and, to a lesser extent, to cytochrome c1.

Regional ultrastructural differences in placental villi in cotyledons of a mature human placenta.

Earlier histological, histochemical, biochemical and autoradiographic studies revealed a marked difference in the morphology and biology of placental villi dependence upon their localization within the materno-fetal circulation units (placentones). The quantitative differences demonstrated characterize the villi in the centres of the circulation units as morphologically and functionally less differentiated than the villi in the periphery. In the present study it should be scrutinized whether these differences can also be proven on the ultramicroscopic level. Small and large villi from both regions (centre and periphery) were studied. Two well-defined syncytial areas, namely syncytium directly overlying a fetal vessel and syncytium over a Langhans cell were studied separately. The following structures were chosen for study and counted by the "grid" method. Microvilli, rough endoplasmic reticulum with parallel channels, vesicular rough endoplasmic reticulum, and the number mitochondria. The ultrastructural studies support the earlier histological, enzyme-histochemical, biochemical and autoradiographic findings, which suggested that the villi in the periphery of the placentones have a higher degree of differentiation (maturation) than those in the centre. There are no qualitative differences between the two regions of the placentone but only quantitative differences which reflect the maturity of the villi.

The interhemal membrane of the spotted hyena: an immunohistochemical reappraisal.

On the basis of specific immunohistochemical staining for vimentin and cytokeratin, we conclude that in the mature placenta of the spotted hyena the interhemal membrane is haemomonochorial rather than endotheliochorial, as in other carnivores. We concur that the intrasyncytial laminae are remnants of the maternal endothelial basal lamina.

Ultrastructural changes in endometriotic implants during the menstrual cycle.

This report, the first ultrastructural study of endometriotic implants, compares the ectopic endometrium in 14 patients with that found in the uterus. The ultrastructural features of most of the glands of the ectopic endometrium resemble those of the corresponding glands lining the uterus. Specific ultrastructural characteristics associated with ovulation, such as giant mitochondria and nuclear channel systems, however, could not be detected in ectopic tissue. Although no pathognomonic electron microscopic characteristics were found in the ectopic endometrium, it was generally possible to assign each sample to a particular phase of the menstrual cycle.

Ultrastructural changes in endometriotic tissue during danazol treatment.

Electron microscopic examinations were carried out on 29 representative endometriotic glands obtained from 16 biopsies. Patients with endometriosis diagnosed by laparoscopy and confirmed by laparoscopic biopsy prior to hormonal treatment received danazol, 800 mg daily. The tissue samples were obtained after 2, 4, and 6 months of usage of the drug to study the ultrastructural effect of this treatment. The glandular epithelium appeared to be arrested in what looked like the late proliferative stage of the normal menstrual cycle. The ciliated cells remained unchanged for the most part. The findings were correlated with possible effects of danazol on the target tissue, but caution is required in the interpretation of ultrastructural effects of steroids until detailed knowledge of the metabolic pathways is available.

Endocrine dependency of endometriosis: an ultrastructural study.

The comparison of the ultrastructural features of endometriotic implants in 96 patients before and after suppressive therapy by danazol showed that the glands of the ectopic endometrium had a wide range of morphologic development. In about one-third of the pretreatment biopsies significantly different ultrastructural patterns were observed in the same specimen, ranging from poorly to highly differentiated endometrial glands. Adequate morphological changes during the menstrual cycle were found in implants only in 14 patients during the proliferative phase, but adequate, homogeneously performed secretory changes were completely missing during the luteal phase. Besides incomplete or delayed secretory changes the majority was proliferative rather than secretory. After 6 months of endocrine suppression laparoscopic biopsies of endometriosis were repeated, and the ultrastructural findings lead to three conclusions. 1. Poorly differentiated endometriotic foci do not respond to danazol. 2. Endometriotic implants consisting of highly differentiated epithelium with adequate cyclic variations respond well to danazol and disappear in nearly 80% of cases. 3. In endometriosis with mixed areas consisting of various degrees of glandular differentiation the hormonal suppression can eliminate endometriotic implants or arrest them at a proliferative stage. If the morphological appearance of the ectopic implants depends not simply upon the endocrine stimulus, but primarily on the degree of differentiation and maturity of the cell, then perhaps cyclic modulation is only a secondary phenomenon, and hormones play only a secondary role in therapy. If this hypothesis is correct, only complete elimination of endocrine influence can cure endometriosis. Transient or incomplete suppression may lead only to partial regression.

Effects of monitoring high-risk pregnancies and intrapartum FHR monitoring on perinates.

A protocol of chronic antepartum surveillance was initiated at the University of Illinois hospitals in 1973 to assess the impact on perinatal mortality. At the same time, a policy of unselected fetal heart rate (FHR) monitoring was initiated to judge the effect on the intrapartum stillbirth rate. The impact of both programs played a significant role in the decline of perinatal mortality rates for infants weighing more than 1 500 g, from 21.1/1 000 births in 1970--1971 to 14.4/1 000 births in the monitored years 1973 and 1974 (p less than 0.02).