The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.     

Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease.     

Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.     

Stage-dependent appearance of sulfhydryl oxidase during spermatogenesis in the testis of rat and hamster. An immunohistochemical study

Sulfhydryl oxidase (SOx), an enzyme that catalyzes the oxidation of sulfhydryl compounds, appears in the spermatogenic cells of rat and hamster testes in a stage-dependent manner. It first appears in pachytene spermatocytes at stage I in both the animal species studied. SOx immunoreactivity is associated with mitochondria of these cells. The fate of such mitochondria is species-dependent. In rat, the immunoreactive mitochondria aggregate during maturation phase and are retained in the residual bodies. Spermatozoa free of SOx are released into the lumen. On the other hand, in hamster, the immunoreactive mitochondria arrange themselves around the midpiece of spermatozoa. In such a case, residual bodies lack SOx. The appearance of SOx coincides with the appearance of LDH-X in the spermatogenic cells. Like many other proteins such as LDH-X, RSA-1 and cytochrome ct, SOx provides yet another example of differential gene activation associated with a developmental process of gametes.     

A cytogenetic study of papaya workers exposed to ethylene dibromide

Ethylene dibromide (EDB) has been shown to be carcinogenic in animal studies and mutagenic in vitro. One cytogenetic study of workers exposed to low levels of EDB for short durations was negative. To test whether exposure to low levels of EDB over long periods caused cytogenetic changes, we have assessed the frequencies of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) in the peripheral blood lymphocytes of 60 men occupationally exposed to EDB. These men worked in papaya-packing plants where EDB was used to fumigate the fruit after harvest to kill fruit-fly larvae. 42 other men who worked at a nearby sugar mill served as controls. The average duration of exposure of the papaya workers was 5 years. 82 full shift personal breathing-zone air samples indicated that the papaya workers were exposed to a geometric mean of 88 ppb of EDB, as an 8-h time weighted average (TWA). Peaks up to 262 ppb were measured. The proposed OSHA 8-h TWA for EDB is 100 ppb, while NIOSH recommends 45 ppb. No differences in SCE levels were found between exposed and nonexposed workers. No differences were found in the total CA frequency between exposed and nonexposed workers. SCE levels were significantly increased in men who smoked cigarettes (p = 0.0001) and in men who smoked marijuana (p = 0.01). CA levels showed a significant increasing trend with age (p = 0.03).     

Highly efficient expression of homologous and heterologous genes in Bacillus megaterium

Summary By developing an efficient transformation system it was possible to reintroduce two different glucose dehydrogenase genes of Bacillus megaterium into this host. These genes were previously cloned, sequenced and expressed in Escherichia coli. Since the expression of one of these genes (gdhA) turned out to be extremely high in B. megaterium, an expression system for genes from closely and distantly related organisms using the controlling region of the gdhA gene was developed.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556

Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms. The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme. The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification. In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity. This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon. The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule. The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate.     

Models for the Generation and Interpretation of Gradients

Source regions for morphogen gradients—organizing regions—can be generated if a local self-enhancing reaction is coupled with a long-ranging reaction that acts antagonistically. Resulting gradients can be translated into patterns of stable gene activities using genes whose products have a positive feedback on the activation on themselves. If several autoregulatory genes compete with each other for activity, cells make an unequivocal choice. Although the signal consists of a smoothly graded distribution, the all-or-nothing response of the cells leads to regions of differently determined cells that are delimited by sharp borders. In some systems, it is not the absolute but the relative level of a gradient that matters. The sequence of head, tentacles, and foot formation in hydra is controlled by a head activation gradient and is an example of this widely used but conceptually rather neglected mode. For subpatterns such as legs and wings, different “compartments” cooperate to produce new signaling substances. Here, morphogen production is restricted to the common borders or where they intersect. The model accounts for the formation of substructures in pairs at the correct positions within the embryo and for the correct orientation and handedness with respect to the main body axes.Many observations in developmental biology can be accounted for by the assumption that graded distributions of substances control cell differentiation in a concentration-dependent manner. Earlier objections against this concept came from two sides. First, the assumption of gradients shifts only the problem as long as no explanation is given of how the gradients are generated in the first place. Second, cells must be extremely sensitive in measuring the absolute concentrations. Only minute concentration differences are expected to exist between adjacent cells. Nevertheless, these must be sufficient to accomplish a reliable and unequivocal selection between alternative pathways.Long before the molecular approach became feasible, we proposed models showing that these features can be realized by molecularly reasonable interactions. Meanwhile, these models found strong support by molecular and genetic observations.