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Biomedical subjects

H Wallace

Publications and source records attributed to H Wallace.

At least 19 recordsLinked to original sources

Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice.

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.

Alpha-Globulins

The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter.

We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box-independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia.

Animals

Specific ablation of thyroid follicle cells in adult transgenic mice.

The coding region of the herpes simplex type 1 virus thymidine kinase gene was coupled to the promoter of the bovine thyroglobulin gene and introduced into the genome of mice. The viral thymidine kinase (HSV1-TK) was expressed mainly in the thyroid glands and testis. Upon treatment of transgenic females with the antiherpetic agent Ganciclovir the thyroid regressed, while the parathyroid gland was unaffected. The number of thyroid follicle cells was greatly reduced after 3 days, and they were completely absent after 7 days of treatment. After 14 days, the levels of circulating T4 and T3 were below the limits of detection, total soluble protein recovered from the thyroid and parathyroid glands together was 10% of the control value, and the level of thyroid HSV1-TK was more than 100-fold lower than that in transgenic controls. Levels of circulating PTH and calcitonin remained normal. At the time of treatment the mice were adults. Thus, the thyroid follicle cells were selectively ablated after normal development with a functional thyroid gland. When treatment with Ganciclovir was terminated after 14 days, no circulating T4 or T3 or other indications of thyroid regeneration were detected for a subsequent period of 90 days. During this time the mice gained weight more slowly than controls, at a rate consistent with the suppression of GH synthesis by thyroid deficiency. The production of mouse major urinary protein (MUP) ceased in the treated mice and was completely restored by the administration of T4. MUP production was not restored by GH, demonstrating that the expression of the Mup genes requires T4 in addition to GH.

Animals

Islands of acetylated histone H4 in polytene chromosomes and their relationship to chromatin packaging and transcriptional activity.

The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labeled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic 'puffs', the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.

Acetylation

Comparative evaluation of New York City and modified Thayer-Martin media for isolation of Neisseria gonorrhoeae.

Commercially manufactured New York City (NYC) medium and modified Thayer-Martin (MTM) medium were compared for their ability to isolate Neisseria gonorrhoeae from clinical specimens. Twenty-seven public health laboratories throughout California evaluated 4,802 specimens collected from patients attending either sexually transmitted disease or family planning clinics. Total of 726 and 737 N. gonorrhoeae isolates were recovered from NYC and MTM medium, respectively. Although less contamination was noted on NYC medium, MTM medium was equivalent to commercially prepared NYC medium for the isolation of N. gonorrhoeae from clinical specimens.

Cervix Uteri

Reliability study of detection of somatic dysfunctions in the cervical spine.

A reliability study was conducted to determine whether palpatory findings on the cervical spine, obtained under normal chiropractic clinical conditions were reproducible. Forty normal freshman chiropractic students underwent static and motion palpation of their cervical spines by three different chiropractors. Intra- and inter-examiner reliability of findings was assessed by concordance statistics (weighted kappa). There was statistically significant agreement of findings for all three examiners only for fixations in the lower cervical spine. There was a total lack of statistical agreement of fixation findings, both intra- and inter-examiner, in the middle cervical area. Agreement of fixation findings was intermediate for the upper cervicals and also for the other palpation parameters that were used (e.g. muscle spasms and pain) throughout the cervical spine. The results generally indicate a relatively high level of replicability of palpatory findings in a normal asymptomatic population by 'typical' chiropractors. The relation of these palpatory findings to vertebral lesions, or subluxations, are unknown.

Cervical Vertebrae

The California Statewide Family Health Education and Training Program 1981-1982.

Through support from the California State Department of Health Services, San Diego State University continued a statewide program in family life education, which consisted of: 1) establishment of a statewide advisory committee representative of many diverse points of view; 2) performance of a study of the teaching of family life education in the ninth and 10th grades of public schools in the state, as well as an examination of the present status of training of teachers; 3) provision of consultation and training programs for local school districts, including community support and implementation training, program development consultation teacher training and a limited number of small block grants to improve local family life education program efforts; and 4) preservice education classes to be conducted at teacher preparation campuses in the California State University system. The paper describes some of the findings of the study of the ninth and 10th grades, as well as the details of methodology of all four components of the statewide program.

California

The response of denervated axolotl arms to delayed amputation.

Forearms of juvenile axolotls can be kept denervated for up to 4 weeks by deflecting brachial nerves to the flank. A more orthodox second denervation prolongs this state up to a total of six weeks. The denervated arms are unable to regenerate for the whole period, but eventually become reinnervated and then regenerate normally. These results and analogous experiments on adult newts prompt a partial retraction and reinterpretation of my previous report on regeneration after prolonged denervation.

Ambystoma mexicanum

I. W. Whimster.

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Dermatology

Duplicated axolotl regenerates.

Three series of palette stage regenerates were prepared by amputating both arms of juvenile axolotls in the mid-forearm, above the elbow, or close to the shoulder. Within each series, excised regenerates were replaced in their original orientation (as a control) or rotated at 90 degrees intervals about the proximodistal axis, or were transplanted to the contralateral arm with identical rotations. Control grafts occasionally caused the formation of a single extra digit. All experimental rotations provoked duplicate or triplicate structures, ranging from accessory digits to extra forearms. Shoulder level grafts were subject to a pronounced correctional derotation and yielded a variable proportion of duplications. Forearm and elbow-level rotations invariably yielded duplications, which therefore result from an axial discrepancy rather than complete axial opposition between graft and host. These results are incompatible with the clockface model of positional information and demand a modification of other current models. The recorded frequency and orientation of duplications suggest that a limb contains at least two polarized transverse axes which cannot be respecified during regeneration. A substantial discrepancy on either axis reduces the normal regulative interaction between graft and host, allowing either of them to regenerate independently.

Ambystoma

Testing the clockface model of amphibian limb regeneration.

Contrary to the predictions of the clockface model, rotating a regenerate by 90 degrees produces duplications identical to those resulting from 180 degrees axial reversals. Exchanging regenerates between arms indicates the presence of 2 determined transverse axes.

Animals

Irradiation inhibits the regeneration of aneurogenic limbs.

The developing arms of axolotl larvae from the 2-digit stage onward and the aneurogenic arms of surgically denervated larvae maintained in parabiosis are able to regenerate after amputation. Such regeneration is uniformly inhibited by local irradiation of the arm, whether innervated or not. This demonstration refutes a recent hypothesis that X-rays interfere with a special activity of nerves required for regeneration, and supports the earlier concept that X-rays act directly on those cells which must proliferate to form the regenerated tissues.

Ambystoma

How x-rays inhibit amphibian limb regeneration.

The effects of an inhibiting dose of 2,000 rad of X-rays on the regenerating limbs of axolotl larvae have been examined in a histological and cytological study. Particular attention was paid to the mitotic indices of normal and irradiated epidermal and blastemal cells. Both the characteristic pattern of epidermal mitotic stimulation which normally follows amputation and the later increase in blastemal mitoses are suppressed by irradiation. In most cells the effects are permanent, but in a small proportion a mitotic delay is induced and upon subsequent division chromosome damage in the form of micronuclei is revealed. Thus irradiated cells which do divide almost certainly die. These results are discussed in relation to other theories of X-ray inhibition of regeneration with particular reference to the view that irradiated cells can be reactivated.

Ambystoma

Dr. G.B. Dowling.

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Dermatology

The cell cycle during amphibian limb regeneration.

The duration of the cell cycle in the blastema of regenerating limbs of axolotls has been measured by means of [3H]thymidine pulse labelling and autoradiography. A chase was required to define the pulse period. An average cell cycle at 20 degrees C takes 53 h, S-phase takes 38 h; including parts of mitosis, G1 is 10 h and G2 is 5 h long. The protracted cycle and S-phase are consonant with the large genome in axolotis and other urodeles. The rapidly growing blastema probably contains a steady population of about 5000 proliferating cells, as there is a regular withdrawal of differentiating cells from the population. The kinds of determination which exist in this population of cells, or are exerted on it, are briefly considered.

Ambystoma