Complete Inactivity Research Articles

CATHEPSIN D AND ITS EFFECTS ON MYOFIBRILLAR PROTEINS: A REVIEW

The molecular and enzymatic properties of the extensively studied enzyme cathepsin D are reviewed and additional information concerning its activity presented. Cathepsin D at pH 5.5 (37°C) degraded several myofibrillar proteins. The most rapidly hydrolyzed included titin and perhaps nebulin, myosin heavy chain, and M and C-proteins. The effects of cathepsin D on myofibrillar structure under these conditions included reduction in A band width, cleared central region in the A band, and dislocation of the Z line. Temperature was found to exert a strong influence on activity of cathepsin D and maximum activity was observed at 45°C with both muscle and hemoglobin substrates. Activity was evident at even higher temperatures and approximately 49% remained at 55°C (hemoglobin assay). Low temperature (i.e., < 15°C) however, has been observed to result in almost complete inactivity of the enzyme. The implications of this information for involvement of cathepsin D in postmortem proteolysis and tenderization were discussed.

Differential loss of neuromuscular connections according to activity level and spinal position of neonatal rabbit soleus motor neurons

We have tested whether the ability of synapses to compete for occupancy of endplates during neuromuscular synapse elimination is affected by differences in the spinal position or in the activity level of the parent motor neuron. To test the role of spinal position, the relative sizes of motor units for motor neurons from middle and extreme (rostral/caudal) positions in the rabbit soleus motor pool were determined at 3 postnatal ages: 4-5 d ("early" ages, when the soleus is heavily polyinnervated), 8-9 d ("intermediate"), and 11-15 d ("late," when the soleus has just reached singly innervated state). Average motor unit sizes from extreme ventral roots were similar to those from middle ventral roots in early-aged soleus muscles but were significantly smaller (by 18-27%) for both intermediate and late muscles. Thus, motor neurons from extreme positions evidently compete less effectively for retention of synapses than those from middle positions. To test the role of differential activity, inactive and active synapses were pitted directly against one another by implanting Silastic plugs laden with tetrodotoxin (TTX) into one of the spinal nerves containing a minority of the soleus motor axons. Differential activity was maintained during a period of extensive synapse loss, from the time of the implant at day 4 or 5 until the intermediate age (day 8-9). Motor unit twitch tensions were subsequently measured to determine the relative number of synapses retained by individual active and inactive motor neurons. The inactivated motor units were on average significantly larger (by more than 50%) than the corresponding group from normal and control-implanted animals. The abnormally large size of inactivated motor units persisted in animals allowed to recover from the TTX block and examined after multiple innervation had disappeared. Hence, the effect of the TTX block cannot be attributed to a simple slowing of synapse elimination specifically among the inactive motor neurons. We conclude that complete presynaptic inactivity improves the chances of survival relative to that for normal activity during synapse elimination in the neonatal rabbit soleus muscle. This difference in competitive ability may contribute to the development of an important characteristic of adult muscles, the correlation between motor unit size and recruitment threshold.

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice.

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.

Secretory patterns of growth hormone during basal periods in man

Human growth hormone (hGH) concentrations in plasma often fall to levels not detectable by RIA. These so-called basal levels prevail during the greater part of the day. Since hGH is involved in the homeostasis of several metabolic processes, it is important to examine its exact plasma concentration and secretory pattern during basal periods. We used an immunoadsorbent technique to extract hGH from large plasma samples to precisely measure basal hGH concentrations and their variation with time. Blood samples (20 mL) were drawn from 12 normal subjects in the fasted and rested state every 15 minutes over a three-hour period. Plasma hGH levels varied over three orders of magnitude (range, 34 to 60,000 pg/mL). During basal periods, episodes of secretory pulses, of moderate sustained secretion, and of complete secretory inactivity occurred. Women had significantly higher overall hGH levels as well as basal hGH levels than men, but no significant sex difference in the pulse frequency during basal periods could be detected in the limited time allotted for study. No convincing relationship was noted between variations in plasma glucose and the secretory pattern of hGH, or vice versa. We conclude that (1) hGH is secreted in an episodic fashion during basal periods. Conceptually, basal and stimulated hGH secretion may be viewed as extremes of a continuous spectrum of pituitary activity, (2) basal hGH levels are lower than heretofore appreciated, (3) the known tendency of women to higher hGH levels is also evident in the basal range, and (4) oscillations in plasma glucose do not affect the microsecretory pattern of hGH, nor are endogenous hGH pulses followed by acute changes in glycemia.

The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences.

We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.

The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences.

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.

Mephenytoin hydroxylation polymorphism: Characterization of the enzymatic deficiency in liver microsomes of poor metabolizers phenotyped in vivo

The rate of 4-hydroxylation and of N-demethylation of S- and R-mephenytoin was determined in liver microsomes of 13 extensive (EM) and two poor (PM) metabolizers of mephenytoin. Detailed kinetic studies were performed in microsomes of eight EMs and the two PMs. Microsomal mephenytoin metabolism in PMs was characterized by an increased Km (150.6 and 180.6 vs. a mean [+/- SD] 37.8 +/- 9.6 mumol/L S-mephenytoin in 8 EMs), a decreased maximum rate of metabolism for S-mephenytoin hydroxylation (0.76 and 0.69 vs 4.85 +/- 1.65 nmol 4-hydroxymephenytoin per milligram protein per hour), and loss of stereoselectivity for the hydroxylation of the R- and S-enantiomers of mephenytoin (R/S ratio: 1.10 and 0.76 vs. 0.11 +/- 0.04 in 13 EMs). The formation of 4-OH-mephenytoin from R-mephenytoin and the demethylation reaction remained unaffected. These results support our hypothesis that the mephenytoin polymorphism is caused by a partial or complete absence or inactivity of a cytochrome P-450 isozyme with high affinity for S-mephenytoin.

Effects of elimination of activity on contractile and histochemical properties of rat soleus muscle

Morphological, contractile, and histochemical properties of rat soleus muscle were studied after 2 or 4 weeks of complete elimination of neuromuscular activity. Inactivity was induced by chronic perfusion of tetrodotoxin (TTX) to the sciatic nerve. Significant reductions in muscle mass and fiber size were found after 2 or 4 weeks of disuse. Correspondingly, the percentage of dark-staining alkaline myosin ATPase fibers was increased from about 20% to 40% after 4 weeks of treatment. The capacity of soleus to generate tension when stimulated through the nerve was significantly impaired at frequencies greater than 20 Hz. Nevertheless, when the curarized muscle was stimulated directly, tension developed at frequencies above 20 Hz relative to peak tension was similar to control values. Absolute tetanic tension was significantly reduced after 2 or 4 weeks of treatment. These reductions could be only partly explained by muscle atrophy, resulting in specific tensions or approximately 55% of control after 2 or 4 weeks of treatment. Measures of the time course of the isometric twitch were found not to be reliable indicators of the contractile speed in TTX-treated soleus. Significant increases in the rate of tetanic tension development, expressed relative to peak tension, and the velocity of unloaded shortening, were seen after 4 weeks of disuse. These results reveal the extent to which virtually complete neuromuscular inactivity leads to chronic deficits in neuromuscular transmission and changes in both the net amount and quality of contractile proteins of rat soleus muscle.

British Food Journal Volume 87 Issue 1 1985

The formulated proposals for this legal principle in the trade battern of the European Community have again appeared in the EEC draft Directive. It has been many years in coming, indicating the extreme difficulties encountered in bringing some sort of harmony in the different laws of Member‐states including those of the United Kingdom, relating to the subject. Over the years there were periods of what appeared to be complete inactivity, when no progress was being made, when consultations were at a stand‐still, but the situation was closely monitored by manufacturers of goods, including food and drink, in the UK and the BFJ published fairly detailed reviews of proposals being considered — in 1979 and 1981; and even as recently as the last few months — in “Consumerism in the Community”, the subject was briefly discussed.

Soldats de la Révolution française en captivité à Szeged.

The soldiers of French Revolution taken prisoners by the Imperial Army in the years 1793-1800 were almost exclusively taken to different parts of Hungary. The reasons, explaining this fact, the arrangement of their transport, the prisoners' emplacement in the country, and the inhabitants' huma feelings and sympathy for the miserable prisoners are discussed by the author. In Szeged the prisoners were emplaced in the fortress. Because of prohibitive measures captivity meant a great torture for them In spite of being forced to complete inactivity the prisoners at last found the way how to make life bearable for themselves. The mercilessness of Fate made it impossible for a great number of them to see again their far away country. Documents bear evidence of 371 French prisoners / died in the years 1793-1800 /, sleeping their last sleep in Szeged. Ladislas PALÂSTI.

DNA sequences and transcription factor interactions of active and inactive forms of mammalian 5 S RNA genes.

Mouse and human 5 S rRNA genes which are, respectively, active and inactive in a human cell-derived transcription system have been characterized. Both contain several base pair substitutions, relative to the gene encoding the prominent 5 S rRNA, indicating that they are variant genes. Both the mouse gene and a Xenopus 5 S rRNA gene are transcribed in systems reconstituted either with the Xenopus 5 S gene-specific factor (IIIA) and other factors from human cells or with the human IIIA equivalent and other factors from amphibian cells. This suggests that structural aspects of the factors relevant to their interactions with each other and with DNA have been conserved and that transcription of mammalian genes is mechanistically similar to that of amphibian genes which employ an internal control region (the site of interaction of IIIA). The latter is also indicated by the demonstrated interaction of the purified Xenopus IIIA factor with both the active mouse and the inactive human gene at intragenic sites corresponding to the internal control site of the amphibian gene; this is observed despite the presence of sequence variations in this region in both of the mammalian genes. Thus, the complete inactivity of the human gene and the lowered activity of the murine gene (relative to the amphibian gene) is not necessarily due to a failure to bind the 5 S gene-specific factor. It is suggested that some control region sequences may be directly or indirectly involved in other factor interactions and the possible roles of specific bases within this region in productive transcription complex formation is discussed. The sequence analyses also suggest alternate termination sequences for class III genes.

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a p I of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the K m for hypoxanthine was 119 μ m, for nicotinic acid was 136 μ m, and for NAD + was 525 μ m.

A long-term hypopigmentary effect of thorium-X on freckled skin.

Sixteen years ago, an area on the forearm of a freckled subject was painted with a solution containing 500 esu of thorium-X per ml. Since then, despite much exposure to solar irradiation, there has been a complete absence of freckles from this area, which stands out in marked contrast to the surrounding skin. Biopsy of the skin from the area was examined by the Dopa technique and by electron microscopy. Melanocytes are present in apparently normal numbers, but in a condition of almost complete inactivity. This depression of melanogenesis is difficult to account for either on the basis of an environmental effect, or of intracellular inhibitory mechanisms.

Effects of DMSO on the structure and function of polytene chromosomes of Chironomus.

Dimethylsulfoxide (DMSO) controlled puff induction and repression (or non-induction) in larval polytene chromosomes of Chironomus tentans were studied for the case of the Balbiani rings (BR). A characteristic reaction pattern, involving BR 1, BR 2, and BR 3, all in salivary gland chromosome IV was found. In vivo exposure of 4th instar larvae (not prepupae) to 10%n DMSO at 18 degrees C first evokes an over-stimulation of BR 3 while DMSO-stimulation of puffing at BR 1 and BR 2 always follows that of BR 3. After removal of the drug, a rapid uniform collapse of all puffs occurs, thus more or less restoring the banding pattern of all previously decondensed chromosome segments. Recovery proceeds as BR's and other puffs reappear. By observing the restoration, one can locate the site from which a BR (puff) originates. BR 2, which is normally the most active non-ribosomal gene locus in untreated larvae, here serves as an example. As the sizes of BR 3, BR 1 and BR 2 change, so do the quantities of the transcriptional products in these gene loci (and vice versa), as estimated electron-microscopically in ultrathin sections and autoradiographically in squash preparations. In autoradiograms, the DMSO-stimulated BRs exhibit the most dense concentration of silver grains and therefore the highest rate of transcriptional activity. In DMSO-repressed BRs (and other puffs) the transcription of the locus specific genes is not completely shut off. In chromosomes from nuclei with high labelling intensities the repressed BRs (and other puffs) always exhibit a low level of 3H-uridine incorporation in vivo. The absence of cytologically visible BR (puff) formation therefore does not necessarily indicate complete transcriptional inactivity. Typically, before the stage of puff formation the 3H-uridine labelling first appears in the interband-like regions.

A selective medium for the isolation of the Acinetobacter genus bacteria.

A selective and differencial medium was developed for the isolation of Acinetobacter genus bacteria. This Acinobacter Agar Medium (p.H + 7.4) contains in grams per litre: thiotone, 10; yeast extract, 3; naC1, 5; saccharose, 10; mannitol, 10; sodium citrate, 0.5; sodium desoxycholate, 0.1; crystal violet, 0.00025; phenol red, 0.04 and agar-agar 15. This medium has the advantage of inhibiting the growth of cocci and Gram-positive bacilli, by the use of sodium citrate and sodium desoxycholate associated with the crystal violet; and of differentiating the Gram-negative bacilli from the Enterobacteriaceae, through the fermentative activity upon the saccharose and/or mannitol, contrasting with the complete inactivity of the Acinetobacter genus bacteria over those substances.

In-vivo myometrial electrical activity in the cyclic mare.

Uterine electromyography was performed by means of chronically implanted surface electrodes in 3 Pony mares during spontaneous oestrous cycles and following luteolysis induced by a prostaglandin analogue (fluprostenol). Three distinct patterns were recognized during the oestrous cycle. (1) During oestrus well defined phases of activity with closely grouped high-amplitude spikes were separated by long periods (10-45 min) of complete inactivity. (2) During dioestrus more diffuse phases of activity with low-amplitude spikes were separated by variable periods of relative inactivity. (3) During luteolysis, short and frequently occurring phases of activity were propagated between the two electrodes on one uterine horn; a similar pattern also occurred between 1 and 3 h after injection of fluprostenol. Peripheral plasma progesterone, but not total inconjugated oestrogen, concentrations were closely related to characteristics of the myographic activity during the cycle. Insemination during oestrus and injection of fluprostenol during dioestrus caused a marked and prolonged increase in myometrial electrical activity. Almost any non-specific environmental stimulus, including entry by palpation of the genital tract per rectum and vaginoscopic examination, but these were of brief duration and the normal resting pattern of activity was quickly re-established after completion of the manipulations.

Neural control of homologous behaviour patterns in two blaberid cockroaches

Activity patterns of motoneurones which innervate spiracular muscles in two blaberid cockroaches, Blaberus discoidalis and Gromphadorhina portentosa, have been monitored during two homologous behaviour patterns: respiratory and non-respiratory tracheal ventilation. Based upon the activity of spiracular motoneurones during these two activities, the abdominal spiracles have been divided into three functional groups: vestigial, respiratory and non-respiratory. In Blaberus discoidalis spiracle 3 is vestigial, spiracles 6, 7, 8 and 10 are respiratory, and spiracles 4, 5 and 9 are non-respiratory. In Gromphadorhina portentosa spiracles 3 and 10 are vestigial, spiracle 4 is non-respiratory and spiracles 5–9 are respiratory. Respiratory spiracles in both species are characterized by activity patterns of their motoneurones during respiratory tracheal ventilation: low frequency firing at irregular intervals during the respiratory pause and a higher frequency burst synchronous with the expiratory abdominal compression. Non-respiratory spiracles are characterized by complete inactivity of their opener motoneurones during respiratory tracheal ventilation. These motoneurones are activated by mechanical stimulation in both species, which simultaneously suppresses activity in respiratory opener motoneurones. In Blaberus discoidalis, there are no differences between activity patterns of respiratory and non-respiratory closer motoneurones. In Gromphadorhina portentosa, not only do respiratory and non-respiratory closer motoneurones have different activity patterns, but the activity pattern of respiratory closer motoneurones is different during respiratory and non-respiratory tracheal ventilation. The functional implications of these several spiracular motoneurone activity patterns are discussed.

The toxic substances control act and in vitro toxicity testing

I want to discuss with you a problem which arose recently and should be of interest to everyone here. There is a bill before Congress-the Toxic Substances Control Act-which might see action in the near future. The reason for concern is the difficulty in seeing it function efficiently once it is activated. To be sure, I am not a spokesman for HEW. The point is that many toxic substances reach the environment, which have not been tested for their toxicity. There may not be enough mice or rats or even investigators around to do the job. But there are other difficulties which became apparent in the last decade and which bring this issue closer to tissue culture research. Some bioassay systems which were developed over the last 50 years have slowly been questioned with regard to their usefulness. As recent as 20 years ago no one took issue, however the results of a test for carcinogenicity turned out, positive or negative, with regard to the interpretation of the data and application of the knowledge gained by regulatory agencies. Recently it has been admitted that a negative test result does not give absolute assurance of complete inactivity and a positive test does not mean that under appropriate conditions of human exposure to the chemical the same results should be expected. In the 40's investigators tackled carcinogenesis testing in whichever way appeared most useful, mostly on mice. Many of these tests are now considered inadequate for extrapolation to man. Today, feeding rats the test chemical is generally accepted but not considered conclusive, so that feeding tests on a second rodent species is recommended before taking action. In this complex situation Congress is looking into the requirements for testing if and when the Toxic Substances Control Act becomes law and how to distribute the cost between the industrial sector and the taxpayer. Not only a staggering expense is involved, but, worse, a lot of time is required. With it comes the question whether industry will have to stop using "questionable" chemicals, now in large-scale production, until convincing "safety data" are available or their use can be continued until definitive decisions regarding safety can be made. If these chemicals are to be tested in animals in ways similar to the human contact, cost estimates may be enormous. For most extensive testing of one chemical, a total of $800,000 was projected; for a chemical requiring less complete testing, $200,000 was the estimate. The question of which tests are necessary for a completely "satisfactory" answer on safety for mankind cannot be answered and therefore should not be asked. Conditions of the bioassay are different from human exposure even if they are mimicked as closely as possible. If an air pollutant is to be inhaled by the test species exactly as by man, we realize that we do not have a proper species at 676

Hormonal Regulation of Larval Diapause

This review examines recent findings about the endocrine basis of larval diapause. Evidence has accumulated to support the view that larval diapause is regulated.by distinctive endocrine interactions. While the lack of the molting hormone, ecdysone, causes the arrest of morphogenesis in both diapausing larvae and pupae, the mecha­ nism leading to the inactivity of the ecdysial glands appears to differ between these life stages. The recent discovery of a juvenile hormone (JH) involvement in the regulation of the larval diapause of two species (24, 144) indicates that at least some species of diapausing larvae retain activity within their endocrine system. This finding has underscored the need to further examine the extent of endocrine activity during larval diapause. For background information, several reviews are available on insect endocrinology (36, 44, 139) and on the physiology and ecology of diapause (35, 87, 121). Hormonal failure and JH regulation are the two principal theories which have been developed to explain larval diapause. The hormonal failure theory was devel­ oped originally to explain the endocrine basis of pupal diapause (136138), and proposes that diapause is instituted when the cerebral neurosecretory (NS) system becomes inactive and the corpora cardiaca stop releasing the ecdysiotropin that is required to stimulate the ecdysial glands to secrete ecdysone. Morphogenesis resumes after the corpora cardiaca renew their release of ecdysiotropin. The dia­ pause state, therefore, is thought to be distinguished by complete inactivity in the endocrine system, including the corpora allata. This theory has been extended to explain the endocrine basis of larval diapause (56, 7 1, 135). Conversely, the JH regulation theory proposes that diapausing larvae, unlike diapausing pupae, retain actively secreting corpora allata and that JH initiates and maintains diapause by regulating the secretion of ecdysiotropin (24, 144). The corpora allata are thought to be controlled by ordinary and NS neurones from the brain. An intermediate titer of JH associated with diapause is believed to arrest morphogenesis by inhibiting

Studies on Metatherian Sex Chromosomes VI. A Third State of an X-linked Gene: Partial Activity for the Paternally Derived Pgk-A Allele in Cultured Fibroblasts of Macropus giganteus and M. parryi

The locus for the glycolytic enzyme phosphoglycerate kinase A (PGK-A) is sex-linked in the eastern grey kangaroo (M. giganteus) and the pretty-face or whip tail wallaby (M. parryi). Females older than the pouch life stage of development do not express their paternally derived allele in some tissues such as blood and liver. This phenomenon is called paternal X inactivation and is characteristic of kangaroos. This distinguishes them from eutherian females, which are mosaics of two kinds of cell brought about by random X inactivation of either the paternally derived or the maternally derived X chromosome. But in skeletal and cardiac muscle the paternally derived allele is weakly expressed. This paper reports data which show that in fibroblast cells cultured from tissue explants of these two species the paternally derived allele is also weakly expressed. This suggests the possibility of a form of mosaicism as in eutherians but with unequal proportions of the two types of cell. Our results show this is not so. Cloning experiments have been performed with cultured cells from three heterozygous M. giganteus females. A total of 49 clones was obtained and each clone was like its parent culture, i.e. there was a strongly active maternally derived allozyme and a weak paternally derived one. Two interpretations of these data are possible. Each cell may have a fully active maternally derived allele and a partly active paternally derived one. Or the clones themselves may be heterogeneous, such that one or the other of the X-linked alleles may switch on or off every few cell generations or less. Since X inactivation is known to be rather stable this latter hypothesis is considered the less likely. Autoradiography shows that the longer arm of the X chromosome in the cultured cells is late-replicating. Thus later replication of the X chromosome in kangaroos does not necessarily mean complete inactivity. Differences in morphology between the two X chromosomes in females are described.