Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).     

Stabilization of ornithine decarboxylase in erythroleukemia cells depleted of ATP

Ornithine decarboxylase activity in Friend erythroleukemia cells decayed with a half-life of 50 minutes after addition of cycloheximide and at a faster rate after addition of spermidine. Incubation with a medium containing dinitrophenol and 2-deoxy-glucose in place of glucose caused ATP depletion and blocked the turnover of ornithine decarboxylase, even after addition of spermidine. Dinitrophenol in the presence of glucose was able to provoke only a slight increase of the half-life of the enzyme. These results suggest that degradation of ornithine decarboxylase in erythroleukemia cells is ATP-dependent.     

Alkaptonuria in an orangutan (Pongo pygmaeus)

Alkaptonuria has been diagnosed in a laboratory born, five year old, female orangutan. In man, this relatively rare amino acid metabolic disorder is characterized by arthritis, pigmentation of cartilage, and darkening of the urine upon standing. The color change is due to oxidation of homogentisic acid not normally found in the urine. The condition in man is a simple Mendelian recessive trait characterized by the absence of homogentisic acid oxidase activity. At two years of age, the affected orangutan was noted to void urine of normal color, which upon standing turned a very dark maroon color. Clinically the animal appeared normal; and the results of a urinalysis, hemogram, and survey radiographs confirmed this evaluation. Nonspecific laboratory determinations used to diagnose alkaptonuria in man were positive. Homogentisic acid in the urine was confirmed by paper chromatography, and a quantitative evaluation was made by a commercial laboratory. The orangutan has shown no clinical change or evidence of ochronosis during the past three years, and quarterly survey radiographs show no evidence of osteoarthropathy. The affected orangutan has half brothers and sisters in the colony but no full sibling. Both parents are young, however, and are being paired in an attempt to produce siblings. It is hoped that additional animals with this metabolic defect will become available for use as potential models for studying this inborn error of metabolism.     

Processing of calling songs by an L-shaped neuron in the prothoracic ganglion of the female cricket, Acheta domesticus

ABSTRACT. An L-shaped auditory intemeuron (LI) has been recorded from extracellularly and intracellularly, and identified morphologically (by Lucifer yellow or cobalt injection) in the prothoracic ganglion of mature female Acheta domesticus. The morphology of the LI is very similar to ascending, prothoracic acoustic interneurons that are most sensitive to higher carrier frequencies in both A. domesticus and other gryllid species. Its terminations in the brain are similar to ascending acoustic interneurons found in other gryllids. The LI neuron is most sensitive to 4–5 kHz model calling songs (CSs), the main carrier frequency of the natural call. Thresholds to high frequencies (8–15 kHz) are 15–20 dB higher. Increasing CS intensities of up to 15 dB above threshold at 4–5 kHz result in increased firing rates by the LI. More than 15 dB increase in intensity causes saturation with little increase in spiking rate until the intensity surpasses 80 dB. In response to 70 dB or higher stimulus intensities, the LI responds to the second and third CS syllables with one or two spikes, pauses, and then produces a burst of nerve impulses with the same or greater latency than for lower intensity stimuli. In response to CS syllables of changing duration (10–30 ms) this neuron responds with a rather constant duration burst of impulses. Syllable periods of the CS stimuli were accurately encoded by the LI. Progressively stronger injection of hyperpolarizing current reduces, and ultimately stops spiking of the LI in response to CS stimuli. More intense stimulation with reduced hyperpolarization shows an initial spike, pause and burst of spikes. Intracellular recording from axonal regions of the neuron shows large spikes, small EPSPs and a developing hyperpolarization through the response to a CS chirp. Inhibitory input to the LI is demonstrated at 4.5, 8 and 16 kHz. This probably explains the specialized response characteristics of the LI which enhanced its encoding of CS syllable period.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

Heterogeneity of antibodies blocking the binding of bungarotoxin to the acetylcholine receptor in myasthenia

Inhibitory effect of myasthenic patients antibodies on alpha-bungarotoxin binding to the human acetylcholine receptor has been demonstrated by radioimmunoassay. By using decamethonium, an acetylcholine agonist, we have shown the existence of two antibody sub-groups reacting with the toxin-binding site: one sub-group is represented by antibodies which block the binding directly, the other by antibodies that inhibit the binding, only in the presence of decamethonium.