Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli.

The inner membrane TET (TetA) protein, which is involved in Tn10-mediated microbial tetracycline resistance, consists of two domains, alpha and beta, both of which are needed for tetracycline resistance and efflux (M.S. Curiale, L.M. McMurry, and S.B. Levy, J. Bacteriol. 157:211-217, 1984). Since tetracycline-sensitive mutants in one domain can partially complement sensitive mutants in the other domain and since some sensitive mutants show dominance over the wild type, a multimeric structure for TET in the membrane had been suggested. We have studied this possibility by using tetA-phoA gene fusions. We fused all but the last 40 base pairs of the tetA gene with the carboxy terminus of the phoA gene for alkaline phosphatase (PhoA), whose activity requires its dimerization in the periplasm. The tetA-phoA fusion protein was under control of the tetracycline-inducible regulatory system for the tetA gene. Induction led to the synthesis of a 78,000-dalton inner membrane protein. Tetracycline resistance was expressed at reduced levels, consistent with the terminal beta domain deletion. Alkaline phosphatase activity was also present, but at low levels, suggesting that some, but not all, of the fusion proteins had their carboxy-terminal ends in the periplasm. When wild-type or mutant TET proteins were present in the same cell with the fusion protein, the tetracycline resistance level was affected (raised or lowered); however, phosphatase activity was reduced only when TET proteins with intact or near-intact beta domains were present. These findings suggest that TET functions as a multimer and that intact beta domains, on TET molecules in the heterologous multimer, either allow fewer PhoA moieties to project into the periplasm or sterically hinder PhoA moieties from dimerizing.     

Characterization of MOPC 315 IgA oligosaccharide processing intermediates

The structures of alpha 1,2-mannose containing partially processed asparagine-linked oligosaccharides on the alpha-chain of MOPC 315 IgA were characterized using specific glycosidases and acetolysis. Man6GlcNAc2, a substrate for a Golgi alpha 1,2-mannosidase, was found to be a single isomeric structure. Likewise, Man7-9GlcNAc2 were single isomers indicating an ordered sequence of removal of alpha 1,2-linked mannose residues on this murine immunoglobulin heavy chain.     

MORPHOLOGY OF ISOLATED RABBIT HEART MUSCLE MITOCHONDRIA AND THE OXIDATION OF EXTRAMITOCHONDRIAL REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.     

Effect of tunicamycin on IgM, IgA, and IgG secretion by mouse plasmacytoma cells

Tunicamycin, an antibiotic that prevents glycosylation of glycoproteins by blocking the formation of N-acetylglucosamine-lipid intermediates, was used to study the importance of glycosylation for the secretion of immunoglobulins by mouse plasmacytoma lines that produce immunoglobulins of different classes. Biosynthetically labeled secreted and intracellular immunoglobulins were measured by immunoprecipitation assays. Tunicamycin, at a concentration of 0.5 mug/ml produced an 81% inhibition of IgM secretion by MOPC 104E plasma cells without significantly affecting the initial rate of synthesis of intracellular IgM. No increase in the intracellular degradation of nonglycosylated IgM could be demonstrated. Tunicamycin also produced a 64% average inhibition of IgA secretion by several mouse IgA-secreting plasmacytoma lines. In contrast, despite inhibiting the incorporation of D-[14C] glucosamine into newly synthesized IgG, tunicamycin only produced a 28% average inhibition of IgG secretion, which was only slightly more than the nonspecific inhibition of secretion of the normally nonglycosylated lambda2 light chains by variant MOPC 315 plasmacytomas. These data indicate that the extent of inhibition of immunoglobulin secretion produced by tunicamycin depends on the immunoglobulin class produced by the plasma cell.     

Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Synchronisation of oestrus of goats treated with progestagen-impregnated intravaginal sponges and PMSG,and reproductive performance following natural mating or A.I. with frozen semen

Oestrus was synchronised in 88 goats using progestagen-impregnated intravaginal sponges (60 mg Repromap; Upjohn) left in place for 17 days, followed by intramuscular injection of PMSG (300 i.u.) at sponge withdrawal. The percentages of does that came into oestrus in 0–24, 25–48, 49–72 and 73–96 h periods were 42, 30.5, 10.5 and 6.5 (cumulative 89.5%). The time of onset of oestrus ranged from 16 to 40 h. Breeding at the synchronised oestrus using intact bucks (24–96 h) or artificial insemination with frozen-thawed semen, at fixed times of 24, 48, 72 and 96 h, resulted in 71% and 81% conceptions respectively. Intrauterine deposition of semen was possible in 31.3%, 62.5%, 87.5% and 6.3% of cases at 24, 48, 72 and 96 h respectively. Twinning percentages were 54.5% and 56% for natural mating and artificial insemination. Kidding was spread over a 14-day period with mean gestation lengths of 149 days for singletons and 147 days for twins. The results indicate that use of progestagen-impregnated sponges with PMSG is an efficient method of synchronisation of oestrus and may result in increased fertility in goats and, if used with A.I. with frozen semen, may contribute to increased breeding efficiency and rate of genetic improvement of goats in Asia.