Free and total thyroid hormones in humans at extreme altitude

Alterations in circulatory levels of total T₄ (TT₄), total T₃ (TT₃), free T₄ (FT₄), free T₃ (FT₃), thyrotropin (TSH) and T₃ uptake (T₃U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized lowlanders, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT₃ and FT₄ occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T₃U was found to be higher at high altitudes. A decline in TSH and hormone binding proteins and an increase in the free moiety of the hormones is indicative of a subtle degree of tissue hyperthyroidism which may be playing an important role in combating the extreme cold and hypoxic environment of high altitudes.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Sub-classification of phage types of Salmonella weltevreden and typing of strains by an extended phage typing scheme

By the use of two adapted phage preparations of Typing phage II the S. weltevreden phage types 4 and 5 could be classified into two sub-types each and phage types 9 and 10 into three sub-types each. The 1094 strains of S. weltevreden could be classified into a total of sixteen phage types including the sub-types.The host range mutants of Typing phage II were distinct from the parent strain. After adaptation to two insensitive strains, one of the new preparations, IIA lost its affinity to some strains which were lysed by the parent phage strain but gained lytic affinity for a few others that were originally insensitive. The second preparation IIB showed an increase in lytic range as expected. Antigenically these preparations were shown to be related but not identical. The possible reasons for serological non-identity of host range mutants with the parent strain have been discussed.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.     

Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain

A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.     

Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein

A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.     

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.