Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

A comparison of the denatured states of alpha-lactalbumin and lysozyme

We have carried out a study of the denaturation of bovine α-lactalbumin by LiClO₄, LiCl, GuCl and urea, using difference spectroscopy, viscometry, polarimetry, and optical rotatory dispersion. These denaturants give rise to three different denatured states (GuCl and urea give the same state), which cannot be related to each other as members of a simple linear progression from the native state to the completely disordered state. The data require that LiClO₄ unfolds one part of the molecule, LiCl another, and GuCl or urea the whole molecule. There are striking parallels between the denaturation behaviour of lactalbumin and earlier observations on hen egg-white lysozyme, a protein with which it is about 40% homologous, and we believe the mass of the data supports the hypothesis, advanced by Browne et al. (1969), that the two proteins have similar backbone conformations.     

Use of the Deane prosthesis in patients on long-term peritoneal dialysis

The Deane peritoneal prosthesis has been used successfully in the treatment of 21 patients with chronic renal failure who were maintained on peritoneal dialysis for periods of up to 20 months. All patients were dialyzed for 24 hours twice weekly. While the prosthesis was still in place, transplantation was carried out in seven patients and laparotomy in three. The prosthesis was also used temporarily whenever a permanent peritoneal catheter (Tenckhoff''s) failed because of infection; it was used until the signs of infection disappeared, then the permanent catheter could be replaced safely. From a total of 1136 dialyses 36 positive cultures were reported. Clinical peritonitis was found on only four occasions.     

A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

The murine aromatic hydrocarbon responsiveness locus: a comparison of receptor levels and several inducible enzyme activities among recombinant inbred lines

The aromatic hydrocarbon responsiveness (Ah) locus has been correlated with genetic differences in the risk of drug toxicity, teratogenesis, chemical carcinogenesis, and mutagenesis. Hepatic cytosolic Ah receptor levels, 2-amino-5-chlorobenzoxazole (zoxazolamine) paralysis time following beta-naphthoflavone treatment and aryl hydrocarbon hydroxylase (AHH3, acetanilide 4-hydroxylase (Ac4H), and NAD(P)H:menadione oxidoreductase (NMOR)4, induction by 3-methylcholanthrene were studied in (a) the progenitors C57BL/6J (Ahb/Ahb) and DBA/2J (Ahd/Ahd) and 25 BXD recombinant inbred lines, (b) the progenitors C57BL/6N and C3H/HeN and 14 B6NXC3N recombinant inbred lines, and (c) the progenitors C57BL/6J and C3H/HeJ and 12 BXH recombinant inbred lines. The Ahb phenotype exhibits greater than 5 femtomole receptor/mg of cytosolic protein, less than or equal to 15 minutes zoxazolamine paralysis time, and twofold to 15-fold induction of these three hepatic enzyme activities; the Ahd phenotype exhibits less than or equal to 2 fmol receptor/mg protein, greater than 15 minutes zoxazolamine paralysis time, and less than 30% induction of these three activities. Among the BXD lines but especially among the B6NXC3N and BXH lines, high frequencies of recombination were found; the phenotype of each of the five parameters did not segregate with the phenotype of each of the other parameters in four or more recombinant lines. This report shows for the first time that AHH induction by 3-methylcholanthrene can occur in the Ahd phenotype mouse. These data underline the complexity of this genetic system when genes from C57BL/6 and DBA/2 are combined and particularly when genes from C57BL/6 and C3H/He inbred mouse strains are combined.     

Silicification of developing internodes in the perennial scouring rush (Equisetum hyemale var. affine)

An electron microprobe (EMP) analysis of silica (SiO₂) deposition in the epidermis of developing internodes of the perennial scouring rush (Equisetum hyemale var. affine) indicates that SiO₂ is first detected in the stomatal apparatus beginning with internode 3, then the epidermal papillae (internode 8), and finally in radial cell walls of the long epidermal cells (internode 10). This process is initiated in the intercalary growth regions at the bases of the elongating internodes. The deposition of SiO₂ in long epidermal cell walls occurs after internodal extension has ceased and should therefore be considered as one of the final stages in internodal differentiation that involves strengthening the cellulosic framework of the cell wall. EMP measurements indicate that SiO₂ in stomata is equivalent to 30% of a pure SiO₂ standard and that SiO₂ in the radial walls of long epidermal cells averages twice that measured on the tangential walls of these same cells. This study supports the view that silicification plays a major role in strengthening the developing perennial scouring rush internodal system and that regulation of this process in this and other species of Equisetum, whose SiO₂ deposition patterns are markedly different, deserves further study.