Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Mammalian artificial chromosome pilot production facility: large-scale isolation of functional satellite DNA-based artificial chromosomes.

BACKGROUND: A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. METHODS: For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line. RESULTS: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional. CONCLUSIONS: By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

The adrenergic inhibitors.

Although most of the centrally and peripherally-acting adrenergic inhibitors have been available for several years, they continue to contribute importantly to antihypertensive therapy. There are remarkably few contraindications to their use. They are useful in hypertension of all grades of severity, and are also valuable in complicated forms of hypertension, such as those associated with renal insufficiency, diabetes mellitus, and chronic obstructive lung disease. They can produce some fairly predictable side effects in patients, but generally do not cause significant metabolic changes. These drugs also seem to be tolerated well by physically active patients. They appear to have desirable effects on cardiac structure. In general, the adrenergic inhibitors cause regression of a left ventricular hypertrophy, which may well be a valuable property, especially in older hypertensive patients.

Doppler echocardiographic assessment of left ventricular systolic and diastolic function in mild hypertension.

Abnormalities in left ventricular (LV) wall thickness and mass have been demonstrated in patients with mild hypertension utilizing M-mode echocardiography. In addition, studies using radionuclide angiography have demonstrated abnormalities in early diastolic LV filling in asymptomatic hypertensive patients with normal ejection fraction and cardiac output. Recently, Doppler recordings of flow velocity in the ascending aorta and through the mitral valve have been shown to provide useful information about LV function. To determine whether flow abnormalities could be detected in patients with mild hypertension, we recorded Doppler aortic and mitral valve flow velocities in 21 men with mild hypertension. Casual systolic blood pressure was 147 +/- 18 mm Hg (mean +/- SD) and diastolic blood pressure was 96 +/- 9 mm Hg. LV mass (310 +/- 75 g) was elevated (i.e., above the 95% normal prediction interval) in 8 of 19 patients who underwent M-mode echocardiography; LV ejection fraction was normal in all patients (mean, 80%). As in previous studies in normal subjects, we found in these hypertensive patients an inverse correlation between age and both aortic peak flow velocity (r = -0.51, p less than 0.05) and transmitral early diastolic peak flow velocity (r = -0.44, p less than 0.05) and a positive relationship between age and mitral valve late diastolic peak flow velocity (r = 0.73, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Disparate relationships between blood pressure and left ventricular mass in patients with and without left ventricular hypertrophy.

The relationship between casual blood pressure and left ventricular (LV) mass has been reported to be fairly weak in hypertensive patients. In this study we analyzed this relationship using noninvasive devices to monitor blood pressure for 24 hours in ambulatory patients and M-mode echocardiography to determine LV mass. Among the 33 patients with hypertension, 21 had echocardiographic LV hypertrophy (LV mass greater than 250 g). Patients with LV hypertrophy did not differ significantly from patients without hypertrophy with respect to age or casual systolic or diastolic blood pressure. The averages of whole-day systolic and diastolic blood pressures were 146 +/- 17 (SD) over 90 +/- 12 and 136 +/- 16 over 89 +/- 12 mm Hg, respectively. The relationship between whole-day average systolic blood pressure and LV mass was significantly positive (r = 0.66, p less than 0.05) in patients without hypertrophy but was not significant in patients with LV hypertrophy (r = -0.24). Similarly, the relationship between whole-day average diastolic blood pressure and LV mass was significantly positive in the former group (r = 0.64, p less than 0.05) but significantly negative in hypertensive patients with LV hypertrophy (r = -0.67, p less than 0.01). Thus, blood pressure correlates positively with LV mass only in patients without cardiac hypertrophy. In hypertensive patients with LV hypertrophy, factors additional to the high blood pressure itself must participate in the regulation of LV mass.

Effect of the calcium channel blocker nitrendipine on left ventricular mass in patients with hypertension.

Reductions in left ventricular (LV) mass have been reported after antihypertensive therapy with certain sympatholytic agents and converting enzyme inhibitors, but little or no improvement has been noted after vasodilator therapy. In this study we evaluated the effect of the calcium channel blocker nitrendipine on echocardiographic LV mass. During a 12-month period, nitrendipine was used as monotherapy in 30 patients and in combination with propranolol or a diuretic in an additional 28 patients. Nitrendipine monotherapy lowered supine blood pressure from 148/97 to 136/83 mm Hg, but LV mass did not change significantly. Supine blood pressure decreased from 155/103 to 134/86 mm Hg in patients receiving combination therapy but, again, changes in LV mass were not significant. These data suggest that nitrendipine is effective in lowering blood pressure, but this is not associated with a significant decrease in LV mass in patients with mild hypertension.

Treatment of hypertension in the elderly.

We have evaluated the effectiveness of antihypertensive therapy for predominant systolic hypertension in 55 patients, aged 61 to 76 years, with untreated systolic blood pressures of at least 160 mm Hg and diastolic blood pressures less than 100 mm Hg. In this retrospective analysis, 41 of the patients had been treated with the centrally acting agent guanabenz (average dose 24 +/- 14 [SD] mg daily) given alone, and 14 had received a combination of guanabenz (17 +/- 10 mg daily) and hydrochlorothiazide (60 +/- 30 mg daily). After six months of therapy, each regimen significantly decreased both systolic and diastolic blood pressures. Moreover, there were no differences between the two treatment regimens in their antihypertensive efficacy, and there was no evidence of orthostatic effects. In both treatment groups, approximately 50% of the patients had excellent therapeutic responses (decrease in supine systolic blood pressure of at least 20 mm Hg). The main side effects of treatment were drowsiness and dry mouth, though these tended to be mild and of short duration. Thus, in predominant systolic hypertension in elderly patients, guanabenz, either alone or in combination with a diuretic, appears to be an effective and well tolerated form of treatment.

Endogenous opioid peptides: do they mediate the acute antihypertensive action of clonidine in humans?

The involvement of endogenous opioid peptides in the antihypertensive action of acutely administered clonidine, a centrally acting adrenergic agonist, was studied in humans. Eight hypertensive subjects received clonidine 0.2 mg orally, naloxone 8 mg i.v. followed by a 0.13 mg/min infusion, and both drugs together on separate days. Clonidine resulted in a significant decrease in mean blood pressure, which was not affected by concomitant treatment with naloxone. Naloxone alone or with clonidine caused significant elevations in plasma aldosterone, not mediated by increased plasma renin activity. Plasma beta-endorphin was not increased after clonidine administration. In humans, the antihypertensive effects of acute clonidine administration do not appear to be mediated by the release or action of endogenous opioids.

Echocardiographic evaluation of left ventricular hypertrophy.

Echocardiography represents an innovative diagnostic method in patients with mild hypertension because it allows evaluation of target organ involvement and, thus, potentially may be of value in determining which patients might most benefit from treatment. In large-scale observations of mild to moderate hypertension, the electrocardiogram and the chest x-ray film each identified approximately 5% of patients as having left ventricular hypertrophy (LVH), whereas echocardiography demonstrated this finding in almost 50%. This high sensitivity also was found in a study of young hypertensive patients (average age 28 years) among whom over 30% had left ventricular muscle wall thicknesses greater than the highest value found in age-matched normotensive controls; additionally, evidence for LVH was documented in a subgroup of young patients with labile hypertension whose blood pressure actually were normal at the time of the echocardiographic procedure. Blood pressure is not the only cause of LVH. Other factors, including heightened activity of the sympathetic nervous system and the renin axis, may be important. Thus, treatment of hypertension with agents such as diuretics or vasodilators that fail to decrease sympathetic activity, or which might even increase it, often fails to produce regression of LVH. In contrast, agents with sympatholytic properties may exhibit beneficial effects on left ventricular muscle mass that are independent of their actions on blood pressure.

Severe hypertension induced by naloxone.

To evaluate the role of endogenous opioid peptides in regulating the blood pressure of hypertensive individuals, we administered the opiate antagonist, naloxone. One individual developed a severe hypertensive response, mean arterial pressure rising from a baseline of 107 mmHg to 147 mmHg 145 min after naloxone injection and infusion. After stopping naloxone, his blood pressure rapidly returned to baseline. Re-challenge with naloxone and clonidine resulted in a modest reduction of blood pressure in contrast to the profound hypotension induced by clonidine alone during a third session. Thus, endogenous opioids appear to regulate blood pressure in some hypertensive patients and opiate antagonists must be administered with caution to these individuals.

Automated ambulatory blood pressure monitoring: a study in age-matched normotensive and hypertensive men.

Noninvasive automated techniques have been used in this study in 29 ambulatory normotensive and hypertensive men to monitor blood pressure during a full 24-hour period. The two groups of men were carefully matched for age. The data were analyzed with respect to possible differences in the circadian pattern of blood pressure, in the variability of blood pressure, and differences in the prevalence of abnormal blood pressures between the two groups. The circadian pattern of blood pressure in hypertensive patients was displayed parallel to that in normotensive control subjects but at a significantly higher level. The difference between daytime and nighttime systolic blood pressure was greater in normotensive than in hypertensive men. However, variability of blood pressure was not different between the two subgroups. Hypertensive men showed on average a greater incidence of abnormal blood pressures than normotensive subjects. However, nine of the normotensive control subjects showed more than 25% of abnormal readings of systolic or diastolic blood pressures during a 24-hour recording period. In contrast, fewer than 25% of abnormal blood pressure readings were found in nine of the hypertensive patients. The data reported here may help to better define the use of ambulatory blood pressure monitoring techniques in the diagnosis of hypertension.

Once-daily administration of acebutolol in treatment of hypertension.

A comparison of once-daily and twice-daily regimens of treatments with a beta-adrenoceptor-blocking agent, acebutolol, was carried out in patients with mild to moderate essential hypertension. Following an initial placebo phase, all patients entered an open 9-week period during which the dose of acebutolol, given on a twice-daily basis to all patients, was titrated to reduce seated diastolic blood pressure to less than 90 mm Hg or to decrease it by at least 10 mm Hg. After a further 4-week maintenance period, patients whose blood pressure had been successfully controlled entered a 3-month double-blind study in which they were randomly divided into three groups: those receiving continuation of twice-daily treatment (n = 47); those receiving once-daily treatment (using the same total daily dose as in the earlier twice-daily phase (n = 97); and those receiving placebo twice daily (n = 48). By the end of the study, 78% of the once-daily treatment group were still under control, a result similar to that of 72% in the twice-daily treatment group. In contrast, only 39% of the placebo patients remained under control. Moreover, the blood pressure in this group was significantly higher than in the other two groups. Acebutolol is an efficacious antihypertensive agent when used on a once-daily basis.

Enhancement of the pressor response to norepinephrine by angiotensin in the conscious rabbit.

The pressor interactions between angiotensin II and norepinephrine were investigated in conscious New Zealand white rabbits receiving a low sodium diet. Angiotensin II was administered continuously by intraperitoneal osmotic pumps in a subpressor dose so as to avoid the potentially confounding effects of experimentally-induced hypertension. Norepinephrine challenges were given as a series of graded intravenous boluses. During the 3 days of study the baseline blood pressure in the angiotensin-treated rabbits (n=10) did not differ from that in controls (n=10) whose intraperitoneal pumps contained only diluent. After 24 hours the systolic and diastolic blood pressure responses to norepinephrine in the angiotensin-treated group were, on average, 45% and 30% higher than in the controls; after 72 hours, they were 46% and 34% higher. Although the pressor amplitudes were increased by angiotensin II, they were not prolonged. Thus, facilitation by the subpressor angiotensin II of the blood pressure responses to norepinephrine did not seem dependent upon alterations in endogenous sympathetic mechanisms or the uptake of norepinephrine; nor could it be explained by sodium retention. It is possible that angiotensin II exhibits its effect by enhancing contractile responsiveness to norepinephrine at the postreceptor level.

Whole-day BP monitoring in ambulatory normotensive men.

Noninvasive automated ambulatory BP monitoring techniques were used to evaluate BP patterns in 34 healthy normotensive men. Daytime BPs (128 +/- 12/80 +/- 7 mm Hg) were significantly higher and nighttime BP averages (109 +/- 11/67 +/- 9 mm Hg) were significantly lower than the casual BPs (119 +/- 13/76 +/- 9 mm Hg) of the subjects studied. On the average, 15.6% of the readings in each tracing showed systolic BPs above 140 mm Hg, and more than 25% of these elevated readings were found in six of the 34 subjects. The average incidence of elevated diastolic BPs (greater than 90 mm Hg) observed during each monitoring period was 14.4%, but six subjects had incidences of more than 25%. The incidence of elevated BP readings was not age related. However, subjects with a family history of hypertension generally had more elevated systolic BPs than those with no family history of hypertension (24% v 9%).