Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis.

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90).

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.

Late diagnosis of Kawasaki disease is associated with haptoglobin phenotype.

BACKGROUND: Kawasaki disease (KD) is an acute febrile illness characterized by multiple clinical and biochemical features of inflammation and the most common complications of coronary artery abnormality (CAA). Haptoglobin (Hp) is an acute-phase protein whose phenotype is known to be involved in coronary artery diseases. In this paper, we report the investigation of the association of Hp phenotype with the formation of CAA in KD. PATIENTS AND METHODS: Forty-seven consecutive patients with clinically diagnosed KD were admitted. Sera were taken before therapy of intravenous immunoglobulins (IVIG) plus aspirin, and levels of serum proteins were measured by a rate immunonephelometer. The echocardiographic criteria for coronary artery abnormality were evaluated during acute or subacute stages. Hp phenotyping was performed by Western immunoblotting. RESULTS: Duration of fever at diagnosis of KD was significantly different between patients with Hp 2-2 (6.4 +/- 1.2 days, n = 25) and with Hp1 allele (Hp 2-1 plus Hp 1-1; 8.8 +/- 3.5 days, n = 22). In contrast, serum levels of Hp between KD patients with Hp2-2 and with Hp1 allele (297 +/- 121 mg dL-1 vs. 330 +/- 101 mg dL-1, respectively) was not significantly different. On the other hand, no patients with Hp 2-2 (0/25) were recognized as having KD in subacute stage. However, 5 out of 20 patients with Hp 2-1 were recognized in subacute stage, and their incidence of CAA was 80.0% (4/5). CONCLUSIONS: Patients with Hp 2-1 have patterns of delayed or incomplete presentation of clinical symptoms. Therefore, the late diagnosis of KD is associated with haptoglobin phenotype.

Amplification and characterization of an inverted repeat from the Chlamydomonas reinhardtii mitochondrial genome.

Based on the nucleotide (nt) sequences of cob and L2a, two oligodeoxyribonucleotides (oligos) were synthesized and used in the polymerase chain reaction (PCR) to amplify the termini of the Chlamydomonas reinhardtii mitochondrial (mt) genome. A 0.8-kb PCR product was detected by agarose-gel electrophoresis when using unligated mt DNA as the template for PCR. This may have indicated the presence of a naturally occurring circular mt DNA molecule that acted as the PCR template. The 0.8-kb DNA could also be amplified from the linear mt DNA via an intramolecular jump during PCR. The sequence data from the 0.8-kb PCR product, and the right 0.6-kb and left 1-kb terminal fragments of the linear mt DNA, along with Southern hybridization analysis, indicated that a 0.49-kb inverted repeat (IR) sequence is present at the right and left termini of the linear mt DNA. The IR contains A+T-rich clusters, as well as numerous short direct repeats (DR) and IR, and might be involved in the recombination, replication and expression of the C. reinhardtii mt genome.

The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease.

The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the alpha-insert) that is present in the cob gene of C. smithii. The alpha-insert, a group I intron (Cs cob.1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob.1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob.1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.

Nucleotide sequence of cloned nad4 (urf4) gene from Chlamydomonas reinhardtii mitochondrial DNA.

Chlamydomonas reinhardtii mitochondrial (mt)DNA was digested with ClaI + HpaI and shotgun cloned into the M13mp19 vector cleaved with AccI + SmaI. One of the recombinant clones, with a 1.8-kb DNA insert, was completely sequenced using the dideoxy chain-termination method. Besides containing part of the cytochrome b (COB)-encoding gene (cob), this DNA fragment encodes subunit 4 of NADH dehydrogenase (NAD4). The deduced amino acid sequence and hydrophilicity plot indicate that NAD4 is highly hydrophobic. The nad4 gene shows a unique preference for certain codons which are also found in other C. reinhardtii mt proteins. Both the genes encoding NAD4 and COB are shown to be transcriptionally active by Northern hybridization. These closely linked genes suggest that RNA-processing events found in vertebrate mt are present in Chlamydomonas mt as well.

Radionuclide fetal imaging: radio-activity in the fetal bladder confirmed by ultrasound after maternal hepato-biliary scanning.

Hepato-biliary scanning with 99mTc-P.G. demonstrated in a jaundiced patient at 31 weeks pregnancy the filling of the fetal bladder 6 h after injection. This finding was confirmed by ultrasound. Fetal urinary bladder activity was seen until 33 h after injection. Fetal hepatic radio-activity could not be displayed although the localisation of the fetal liver was marked by ultrasound.

Hepatobiliary scanning with 99mTc-pyridoxylidene glutamate. A retrospective study investigating the criteria for differentiation between intrahepatic and extrahepatic obstruction.

In 70 99mTc-pyridoxylidene glutamate (PG) studies with verified diagnoses, the following scan patterns were found. (1) Normal: within 30 min of PG injection the scan reveals the liver, hepatic ducts, common bile duct, gallbladder and flow to the intestine; after 2 h the liver had a higher concentration of activity than the hepatic ducts or the common bile duct. (2) Complete extrahepatic obstruction: no hepatic excretion to the intestine is observed 18-24 h after PG injection, nor is activity observed in the hepatic ducts, common bile duct and gallbladder. (3) Incomplete extrahepatic obstruction: intestinal activity is observed within 18-24 h of PG injection; after 2 h the concentration of activity in the hepatic ducts or the common bile duct exceeds that in the liver (regardless whether activity is or is not demonstrated in the gallbladder). (4) No extrahepatic obstruction: serum bilirubin normal or increased; intestinal activity is observed within 18-24 h after PG injection, and activity is demonstrable during this period somewhere in the hepatic ducts, the common bile duct or the gallbladder; after 2 h the concentration of biliary activity should not exceed that in the liver. (5) If excretion to the intestine is observed within 18-24 h of PG injection without demonstrable activity in the hepatic ducts, common bile duct or gallbladder, then it is impossible to differentiate between (3) and (4).

Sensitivity of ultrasound in the detection of biliary tract obstruction.

In 28 patient with varying degrees of biliary obstruction, the ultrasonic findings were correlated with the serum bilirubin levels and with the size of the bile ducts as measured by percutaneous transhepatic cholangiography (PTC). With ultrasound, study of the intrahepatic ducts achieved the greatest accuracy (71%); examination of common bile ducts and the gallbladder were found to be less sensitive (54 and 53%). Ultrasound proved to be very accurate in biliary obstruction, when the serum bilirubin was above 4.8 mg/100 ml (80 mumol/1).