Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

Ventilatory responses to specific CNS hypoxia in sleeping dogs.

Our study was concerned with the effect of brain hypoxia on cardiorespiratory control in the sleeping dog. Eleven unanesthetized dogs were studied; seven were prepared for vascular isolation and extracorporeal perfusion of the carotid body to assess the effects of systemic [and, therefore, central nervous system (CNS)] hypoxia (arterial PO(2) = 52, 45, and 38 Torr) in the presence of a normocapnic, normoxic, and normohydric carotid body during non-rapid eye movement sleep. A lack of ventilatory response to systemic boluses of sodium cyanide during carotid body perfusion demonstrated isolation of the perfused carotid body and lack of other significant peripheral chemosensitivity. Four additional dogs were carotid body denervated and exposed to whole body hypoxia for comparison. In the sleeping dog with an intact and perfused carotid body exposed to specific CNS hypoxia, we found the following. 1) CNS hypoxia for 5-25 min resulted in modest but significant hyperventilation and hypocapnia (minute ventilation increased 29 +/- 7% at arterial PO(2) = 38 Torr); carotid body-denervated dogs showed no ventilatory response to hypoxia. 2) The hyperventilation was caused by increased breathing frequency. 3) The hyperventilatory response developed rapidly (<30 s). 4) Most dogs maintained hyperventilation for up to 25 min of hypoxic exposure. 5) There were no significant changes in blood pressure or heart rate. We conclude that specific CNS hypoxia, in the presence of an intact carotid body maintained normoxic and normocapnic, does not depress and usually stimulates breathing during non-rapid eye movement sleep. The rapidity of the response suggests a chemoreflex meditated by hypoxia-sensitive respiratory-related neurons in the CNS.     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.     

Weak interaction of spectrin with phosphatidylcholine-phosphatidylserine multilayers: a 2H and 31P NMR study

Spectrin from human erythrocytes binds to bilayer dispersions of both DMPC and DMPS:DMPC (1:1, w/w). However, no effect of bound spectrin on the conformation of the lipid head groups, as measured from the deuterium quadrupolar splittings of DMPC or DMPS specifically deuterated in the polar head groups, was detected in 1:1 mixtures of the two lipids containing either deuterated DMPC or DMPS. Neither the phase transition of the DMPS:DMPC mixtures, nor the spin-lattice relaxation time (T1) of the deuterated DMPS head group, was affected by spectrin. These results argue against any strong interaction of spectrin with phosphatidylserine and rule out the possibility that spectrin is responsible for the maintenance of PS in the inner monolayer of the erythrocyte membrane during the whole life-span of this cell.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Bone marrow derived macrophages have polyamine and ectoenzyme phenotypes distinct from resident macrophages

Several prototype macrophage (MO) populations were compared for differences in ectoenzyme phenotype and polyamine content. Resident peritoneal MO and Corynebacterium parvum (CP)-activated peritoneal MO expressed unique ectoenzyme phenotypes, while bone marrow derived MO (BMDMO), obtained from stem cells after 7 days in culture with colony stimulating factor, and thioglycollate (TG)-elicited peritoneal MO exhibited a similar ectoenzyme phenotype. All of the MO populations, however, differed in polyamine accumulation patterns. These results suggest that ectoenzyme phenotypes do not serve as completely selective markers of MO differentiation. Moreover, BMDMO do not resemble steady state tissue peritoneal MO but appear to resemble inflammatory MO in several respects. Therefore activated BMDMO do not appear to provide an accurate model system for their continued use in studies to characterize the development of resident tissue MO.     

Effects of altered CSF [H+] on ventilatory responses to exercise in the awake goat

We investigated the effects of selective large changes in the acid-base environment of medullary chemoreceptors on the control of exercise hyperpnea in unanesthetized goats. Four intact and two carotid body-denervated goats underwent cisternal perfusion with mock cerebrospinal fluid (CSF) of markedly varying [HCO-3] (CSF [H+] = 21-95 neq/l; pH 7.68-7.02) until a new steady state of alveolar hypo- or hyperventilation was reached [arterial PCO2 (PaCO2) = 31-54 Torr]. Perfusion continued as the goats completed two levels of steady-state treadmill walking [2 to 4-fold increase in CO2 production (VCO2)]. With normal acid-base status in CSF, goats usually hyperventilated slightly from rest through exercise (-3 Torr PaCO2, rest to VCO2 = 1.1 l/min). Changing CSF perfusate [H+] changed the level of resting PaCO2 (+6 and -4 Torr), but with few exceptions, the regulation of PaCO2 during exercise (delta PaCO2/delta VCO2) remained similar regardless of the new ventilatory steady state imposed by changing CSF [H+]. Thus the gain (slope) of the ventilatory response to exercise (ratio of change in alveolar ventilation to change in VCO2) must have increased approximately 15% with decreased resting PaCO2 (acidic CSF) and decreased approximately 9% with increased resting PaCO2 (alkaline CSF). A similar effect of CSF [H+] on resting PaCO2 and on delta PaCO2/VCO2 during exercise also occurred in two carotid body-denervated goats. Our results show that alteration of the gain of the ventilatory response to exercise occurs on acute alterations in resting PaCO2 set point (via changing CSF [H+]) and that the primary stimuli to exercise hyperpnea can operate independently of central or peripheral chemoreception.     

High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.