Ice-nucleating bacteria from the guts of two sub-antarctic beetles, hydromedion sparsutum and perimylops antarcticus (Perimylopidae)

The site of ice nucleation in the freeze-tolerant, sub-Antarctic beetle Hydromedion sparsutum has been investigated. Ice+ bacteria, active at above -2.0 degrees C, were isolated from the guts of beetles and identified as a fluorescent Pseudomonas species. Other possible sites of nucleation, including the hemolymph, were examined but had a lower activity. Ice+ bacteria were isolated from mixed populations, isolated from the guts of adult beetles, and grown on nutrient agar plates and in nutrient broth. Nucleation activity of the broth culture peaked after only 2 days although the number of live cells continued to increase until day 6. These cultures were used to determine the maximum nucleation activity of a bacterial suspension in sterile distilled water (-3.4 degrees C) and the dilution factor required to cause a 50% reduction in activity (10(4)). The original bacterial suspension had an absorbance of 0.5 measured at 660 nm and contained 6 x 10(11) bacteria per milliliter. From this it is estimated that only 1 in 10(6) bacteria possessed the highest levels of ice-nucleating activity. Other insect species, including Perimylops antarcticus, which are found in habitats similar to that of H. sparsutum, were examined for the presence of ice+ bacteria. All contained ice-nucleating bacteria in their guts but with a lower level of activity than in H. sparsutum. Copyright 1999 Academic Press.     

Effects of structure on alpha C-H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation.

The bond dissociation enthalpies (BDE) of all of the amino acid residues, modeled by HC(O)NHCH(R)C(O)NH(2) (PH(res)), were determined at the B3LYP/6-31G//B3LYP/6-31G level, coupled with isodesmic reactions. The results for neutral side chains with phi, psi angles approximately 180 degrees, approximately 180 degrees in ascending order, to an expected accuracy of +/-10 kJ mol(-)(1), are Asn 326; cystine 330; Asp 332; Gln 334; Trp 337; Arg 340; Lys 340; Met 343; His 344; Phe 344; Tyr 344; Leu 344; Ala 345; Cys 346; Ser 349; Gly 350; Ile 351; Val 352; Glu 354; Thr 357; Pro-cis 358; Pro-trans 369. BDEs calculated at the ROMP2/6-31G//B3LYP/6-31G level exhibit the same trends but are approximately 7 kJ mol(-)(1) higher. All BDEs are smaller than those of typical secondary or tertiary C-H bonds due to the phenomenon of captodative stabilization. The stabilization is reduced by changes in the phi,psi angles. As a result the BDEs increase by about 10 kJ mol(-)(1) in beta-sheet and 40 kJ mol(-)(1) in alpha-helical environments, respectively. In effect the alpha C-H BDEs can be "tuned" from about 345 to 400 kJ mol(-)(1) by adjusting the local environment. Some very significant effects of this are seen in the current literature on H-transfer processes in enzyme mechanisms and in oxidative damage to proteins. These observations are discussed in terms of the findings of the present study.     

Diterpenes from the leaves of Croton zambesicus

Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one; isopimara-7,15-dien-3beta-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, beta-sitosterol, alpha-amyrin and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these compounds has been evaluated on cancer and non-cancer cell-lines.     

Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.     

Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement

Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha.     

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.     

Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.     

The importance of lattice defects in katanin-mediated microtubule severing in vitro

The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.