HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.     

Fine structure of the eggs of blowflies Aldrichina grahami and Chrysomya pacifica (Diptera: Calliphoridae)

We report here the fine structure of the eggs of blowflies Aldrichina grahami (Aldrich) and Chrysomya pacifica Kurahashi. For A. grahami, the plastron is wide and extends to almost the entire length of the eggs. The plastron near the micropyle is truncated. The polygonal patterns of chorionic sculpture bear a distinct swollen boundary. Regarding C. pacifica, the plastron is narrow and extends to almost the entire length of the eggs. The plastron near the micropyle bifurcates to a Y-shape, but the arms of the 'Y' are short. Information presented herein allows some distinctive features to differentiate among eggs of blowfly species.     

Effect of lung-protective ventilation on severe Pseudomonas aeruginosa pneumonia and sepsis in rats

Pneumonia caused by Pseudomonas aeruginosa carries a high rate of morbidity and mortality. A lung-protective strategy using low tidal volume (V(T)) ventilation for acute lung injury improves patient outcomes. The goal of this study was to determine whether low V(T) ventilation has similar utility in severe P. aeruginosa infection. A cytotoxic P. aeruginosa strain, PA103, was instilled into the left lung of rats anesthetized with pentobarbital. The lung-protective effect of low V(T) (6 ml/kg) with or without high positive end-expiratory pressure (PEEP, 10 or 3 cmH(2)O) was then compared with high V(T) with low PEEP ventilation (V(T) 12 ml/kg, PEEP 3 cmH(2)O). Severe lung injury and septic shock was induced. Although ventilatory mode had little effect on the involved lung or septic physiology, injury to noninvolved regions was attenuated by low V(T) ventilation as indicated by the wet-to-dry weight ratio (W/D; 6.13 +/- 0.78 vs. 3.78 +/- 0.26, respectively) and confirmed by histopathological examinations. High PEEP did not yield a significant protective effect (W/D, 4.03 +/- 0.32) but, rather, caused overdistension of noninvolved lungs. Bronchoalveolar lavage revealed higher concentrations of TNF-alpha in the fluid of noninvolved lung undergoing high V(T) ventilation compared with those animals receiving low V(T). We conclude that low V(T) ventilation is protective in noninvolved regions and that the application of high PEEP attenuated the beneficial effects of low V(T) ventilation, at least short term. Furthermore, low V(T) ventilation cannot protect the involved lung, and high PEEP did not significantly alter lung injury over a short time course.     

Role of intracellular Ca2+ in endothelium-dependent contraction and relaxation of rabbit intrapulmonary arteries

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.     

A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI+] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

The yeast prion [PSI⁺] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI⁺] propagation, however the structure-and-function relationship of Hsp104 for [PSI⁺] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI⁺] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI⁺] propagation. However, nine mutants were impaired exclusively for [PSI⁺], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI⁺] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI⁺] prion aggregates and that the lateral channel plays a role specific to [PSI⁺] prion propagation.Key Words: Hsp104, reverse genetics, hexamer, nonsense suppression, yeast prion [PSI⁺], thermotolerance     

Genetic evidence for possible interaction between a ribonucleic acid polymerase subunit and the spo0C gene product of Bacillus subtilis.

Spontaneous rifampin-resistant mutants (9V Rifr) were isolated from a mutant strain of Bacillus subtilis, 9V, which has a spo0C mutation. Whereas 90% of the 9V Rifr double mutants maintained the Spo0C phenotype (Spo- Abs +/-), the remaining 10% had the Spo0A phenotype (Spo- Abs-). The latter mutants, termed 9V Rifr Spo- Abs-, were revealed to have other Spo0A characters, such as reduced transformability, higher sensitivity to phage phi 2, and reduced frequency of lysogenization by phage phi 105. The rif mutation of these 9V Rifr Spo- Abs- strains was mapped near the cysA locus. The phenotype of the Rifr transformants of strain 9V by deoxyribonucleic acid derived from these 9V Rifr Spo- Abs- strains was Spo0A, and that of the Rifr transformants of strain 168 was Spo+ Abs+. The ribonucleic acid polymerase of the 9V Rifr Spo- Abs- strains was shown to be resistant to rifampin.     

Alteration of the LIS1 gene in Japanese patients with isolated lissencephaly sequence or Miller-Dieker syndrome

LIS1 is a genetic entity that is responsible for lissencephaly. Previously we have reported isolated lissencephaly sequence(ILS) in a Japanese patient carrying a balanced chromosomal translocation that disrupted the LIS1 gene. We examined mutations of LIS1 in 12 additional Japanese patients, 8 of them with ILS and 4 with Miller-Dieker syndrome (MDS). Fluorescence in situ hybridization (FISH) analysis disclosed deletions of part of the LIS1 gene or of the chromosomal region surrounding it in three of the ILS cases and in three of the MDS cases. In one of the remaining five ILS cases, SSCP analysis and subsequent sequence analysis identified a 1-bp deletion in exon IV, which can be expected to result in premature termination of the gene product. Our results indicate that in Japan, as elsewhere, abnormality of the LIS1 gene is a common cause of MDS/ILS. Received: 20 April 1998 / Accepted: 28 July 1998