Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase,a multifunctional polyketide biosynthetic enzyme,by free coenzyme A

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.     

Prediction of an HMG-box fold in the C-terminal domain of histone H1: insights into its role in DNA condensation

In eukaryotes, histone H1 promotes the organization of polynucleosome filaments into chromatin fibers, thus contributing to the formation of an important structural framework responsible for various DNA transaction processes. The H1 protein consists of a short N-terminal "nose," a central globular domain, and a highly basic C-terminal domain. Structure prediction of the C-terminal domain using fold recognition methods reveals the presence of an HMG-box-like fold. We recently showed by extensive site-directed and deletion mutagenesis studies that a 34 amino acid segment encompassing the three S/TPKK motifs, within the C-terminal domain, is responsible for DNA condensing properties of H1. The position of these motifs in the predicted structure corresponds exactly to the DNA-binding segments of HMG-box-containing proteins such as Lef-1 and SRY. Previous analyses have suggested that histone H1 is likely to bend DNA bound to the C-terminal domain, directing the path of linker DNA in chromatin. Prediction of the structure of this domain provides a framework for understanding the higher order of chromatin organization.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

Apoptosis: Identification of dying cells

Cell death is usually classified into two broad categories: apoptosis and necrosis. Necrosis is a passive, catabolic process, always pathological, that represents a cell's response to extreme accidental or toxic insults. Apoptosis, in contrast, occurs under normal physiological conditions and is an active process requiring energy. However, apoptosis can also be elicited in a pathological way by toxic injury or during disease processes. In these nonphysiological conditions, both types of cell death can be encountered following the same initial insult and the balance between death by apoptosis and by necrosis appears to depend upon the intensity of the injury and the level of available intracellular ATP. It is important, however, to discriminate between apoptosis and necrosis in pathological conditions, as therapeutic intervention could be considered in apoptotic cell death with putative new pharmacological agents aimed at interfering with the key molecular events involved. In most cases, none of the current laboratory techniques used alone allows for unambiguous identification of apoptotic cells. Some of the most common methods based on morphology, biochemistry, and plasma membrane changes are discussed in terms of specificity and possible sources of error in data interpretation. As a rule, classification of cell death in a given model should always include morphological examination coupled with at least one of the other assays.     

Sperm ultrastructure, morphometry, and abnormal morphology in American black bears (Ursus americanus)

The objective of this study was to describe sperm ultrastructure, morphometry, and abnormal morphology in American black bears. Electroejaculation was successful in 53.8% (7/13) of the attempts, but urine contamination was common. Epididymal sperm samples were also obtained from five bears. Sperm had a paddle-like head shape and the ultrastructure was similar to that of most other mammals. The most striking particularity of black bear sperm ultrastructure was a tightening of the nucleus in the equatorial region. Although the differences were not significant in all bears, the overall decrease in sperm nucleus dimensions during transport from the caput epididymis to the cauda suggested increasing compaction of the nucleus during maturation. For ejaculated sperm, nucleus length, width, and base width were 4.9, 3.7, and 1.8 μm, respectively, whereas sperm head length, width, and base width were 6.6, 4.8, and 2.3 μm, and midpiece, tail (including midpiece), and total sperm lengths were 9.8, 68.8, and 75.3 μm. Evaluation of sperm cytoplasmic droplets in the epididymis revealed that proximal droplets start migrating toward a distal position in the caput epididymis and that the process was mostly completed by the time sperm reached the cauda epididymis. The proportion of morphologically normal sperm in the ejaculate was 35.6%; the most prevalent sperm defects were distal cytoplasmic droplets and bent/coiled tails. The morphology of abnormal sperm and the underlying ultrastructural defects were similar to that in other large domestic animals thus suggesting similar underlying pathogenesis of specific sperm defects and similar effects on fertility.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

Question 3: The Problem of Macromolecular Sequences: The Forgotten Stumbling Block

The Author agrees in principle with the question/statement, but states also that an important qualification is needed within this question. In fact, it is not possible by the bottom up approach to find the conditions for the synthesis of our actual proteins—lysozyme, chymotrypsin or the like—however it is possible to show experimentally that co-oligopeptides chains of that length can be produced by prebiotic reactions. Considering such a synthesis, it is important to recall that proteins—and nucleic acids—are not simply polymers, but are co-polymers, and the kinetics and thermodynamics attending the synthesis of copolymers poses stringent constraints for the biogenesis and growth of specific sequences. Such constraints are examined and discussed. Presented at: International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Shaping mitotic chromosomes: From classical concepts to molecular mechanisms

How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss – in light of these recent insights – the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra‐chromosomal linkages by condensin complexes in the context of cohesin‐mediated inter‐chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod‐shaped metaphase chromosomes ready for their segregation to the cell poles.