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Biomedical subjects

J C Probst

Publications and source records attributed to J C Probst.

At least 37 records · Page 2Linked to original sources
PubMed1996

Elevated messenger RNA levels after antisense oligodeoxynucleotide treatment in vitro and in vivo.

Antisense oligodeoxynucleotides are being increasingly employed in various fields of biological research. Not only has the antisense mediated inhibition of gene expression become a valuable experimental tool, the synthetic oligodeoxynucleotides are also considered to be potential therapeutic drugs. Despite the huge body of literature regarding antisense treatment, there is little information on the mode of action. Currently, the main view is that antisense oligodeoxynucleotides bind to the target mRNA and thus trigger RNase H-directed cleavage of the newly formed RNA-DNA duplex molecules. Here we present data from in vitro and in vivo experiments showing that antisense oligodeoxynucleotide treatment does not necessarily lead to a reduction of the corresponding mRNA levels. In fact, mRNA levels can be clearly elevated. These results favor the view that antisense oligodeoxynucleotides, presumably in a large number of neurobiological applications, function via hybrid arrested translation rather than RNase H-driven mRNA cleavage.

Animals↗
PubMed1996

Human intestinal trefoil factor is expressed in human hypothalamus and pituitary: evidence for a novel neuropeptide.

Human intestinal trefoil factor, hITF, a secretory polypeptide found mainly in the human gastrointestinal tract, is a member of the newly characterized trefoil factor or P-domain peptide family representing putative growth factors. Here we describe the identification of this gut peptide in the human brain and pituitary. With reverse transcriptase polymerase chain reaction, we were able to isolate and clone the transcript from human hypothalamus. An antibody generated against a synthetic peptide derived from the carboxyl terminus of hITF was used for immunohistochemical studies of appropriate tissue sections. Neurons expressing hITF were identified in two magnocellular hypothalamic nuclei, the paraventricular and periventricular nuclei. hITF polypeptide was also observed in Herring bodies of the neurohypophysis and in secretory cells of the adenohypophysis. Double immunostaining with antigrowth hormone antibody showed partial coexistence in a selected subpopulation of adenohypophysial cells. Localization of hITF in the hypothalamo-neurohypophysial system may suggest a modulatory action on the classical magnocellular nonapeptides vasopressin and oxytocin, and further indicates an adenohypophysial importance of this peptide. It is likely that hITF represents a novel neuropeptide of yet unknown function.

Adult↗
PubMed1996

Norplant prescribing in family practice.

BACKGROUND: This study describes the Norplant prescribing experience, training, and attitudes of South Carolina family practice and general practice (FP/GP) physicians. METHODS: A survey was mailed to all FP/GP physicians licensed in South Carolina, with two follow-up mailings to nonrespondents. RESULTS: Responses were received from 520 physicians, representing 43% of all licensed FP/GP physicians and 73% of all board-certified FP physicians. Norplant had been inserted by 39% of the respondents, and fewer than 10% reported encountering complications during the insertion process. Norplant removal was reported by 135 physicians, averaging 4.4 removals per year. Complications during removal were reported by 52% of physicians, with the most common problem being difficulty finding the capsules. Some degree of training in Norplant insertion was reported by 82% of physicians; 69% reported having had some training, formal or informal, in removal; but only 57% reported having received formal training either during residency or at a workshop. Training reduced insertion time but did not affect removal time or the number of complications encountered. Formal training was more likely to prepare physicians to successfully manage procedural complications. CONCLUSIONS: FP/GP physicians are important providers of Norplant. Improved training is needed to ensure that insertion is performed properly and to disseminate effective removal techniques.

Adult↗
PubMed1995

Chronic infusion of a CRH1 receptor antisense oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-related behavior in socially defeated rats.

We studied the role of central amygdala CRH receptors in behavioral responses to an anxiogenic stimulus. An antisense oligodeoxynucleotide corresponding to the rat CRH1 receptor mRNA was infused chronically into the central amygdaloid nucleus of male rats via osmotic minipumps (0.25 micrograms/0.5 microliters/h). Control groups received infusions of either a scrambled sequence oligodeoxynucleotide or vehicle. On the 4th day of treatment, rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Antisense oligodeoxynucleotide-treated rats spent significantly more time exploring the open arms of the plus-maze than scrambled sequence- and vehicle-treated animals, both of which did not differ from each other. The social discrimination test, on the other hand, revealed no difference in juvenile recognition abilities among the treatment groups. Using in situ hybridization and receptor autoradiography, we were not able to detect clear signals of CRH1 receptor mRNA and CRH binding sites in the central amygdaloid nucleus of either group, confirming the reportedly low expression and density of CRH receptors in this brain area. The present data support the view that CRH receptors in the central nucleus of the amygdala are involved in the mediation and expression of anxiety-related behavior, but simultaneously raise questions as to the mechanisms of antisense oligodeoxynucleotide action.

Amygdala↗
PubMed1995

Molecular and cellular analysis of rP1.B in the rat hypothalamus: in situ hybridization and immunohistochemistry of a new P-domain neuropeptide.

P-domain peptides, a new family of secretory polypeptides, have been identified mainly in the gastroenteropancreatic tract of humans, rodents, and amphibians as well as in amphibian skin. In the present study, with PCR and RNA analysis a transcript has been discovered in rat brain termed rP1.B. The deduced polypeptide consists of a single P-domain and its amino acid sequence matches that of rat intestinal trefoil factor (rITF). Thus far, rP1.B is the only P-domain peptide expressed in neuronal cells of the CNS. Immunostained magnocellular perikarya were visible in the paraventricular, supraoptic and periventricular nuclei. Parvocellular rP1.B neurons were found in the arcuate nucleus. Additionally, specific hybridization signals with radiolabeled transcripts were observed in the same regions. rP1.B in the rat hypothalamus may be involved in the control of hypothalamo-hypophysial functions.

Amino Acid Sequence↗
PubMed1995

Antisense oligodeoxynucleotide complementary to oxytocin mRNA blocks lactation in rats.

The posterior lobe peptide oxytocin (OT) is known to control lactation and parturition, as well as maternal and sexual behavior. An antisense oligodeoxynucleotide (ODN) directed against the mRNA of OT was injected intracerebroventricularly 6 times in 12 hour intervals to manipulate the transcriptional message of OT in lactating rats. OT immunoreactivity in magnocellular hypothalamic nuclei and in the posterior lobe of the pituitary was reduced in antisense treated animals in comparison to ODN with scrambled base composition and vehicle controls. This decline in OT levels was associated with a decrease of pup weight. Our results demonstrate that central infusions of antisense ODN significantly reduce OT expression in vivo.

Animals↗
PubMed1995

V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behavior in rats.

To develop and validate a vasopressin (AVP) receptor knockdown strategy, we infused an antisense oligodeoxynucleotide to the V1 subtype mRNA into the septum of male rats with osmotic minipumps and measured behavioral, cellular and molecular parameters. Compared to vehicle and scrambled-sequence oligo controls, chronic antisense administration for up to 4 d diminished the ability of the animals to distinguish a previously exposed juvenile from a novel one and to respond to exogenous AVP (1 ng/5 microliters, intracerebroventricular) with an improved social memory. Furthermore, anxiety-related behavior was reduced. As measured in the behaviorally tested rats, antisense treatment resulted in a reduced binding of radiolabeled AVP in the septum, but not in other limbic brain areas (receptor autoradiography), and an increased amount of V1 receptor mRNA (reverse transcriptase PCR), indicating translational arrest and ongoing transcriptional activity. In sense oligo-treated rats, on the other hand, both the social and the anxiety-related behavior scores lay between levels obtained in control and antisense-treated animals. These sense-treated rats showed a slightly reduced V1 receptor density in the septum and reduced receptor mRNA levels, indicating hybridization of the sense oligo to the DNA. The data show the potential of antisense targeting to further reveal relationships between local gene expression, neuropeptide-receptor interactions in distinct brain areas, and behavioral performance.

Analysis of Variance↗
PubMed1995

After-hours telephone calls in a family practice residency: volume, seriousness, and patient satisfaction.

BACKGROUND: After-hours calls are common in primary care physicians' practices. Calls may be unnecessary from the physician's perspective, but patients may have a different concept of the importance of reaching their physician immediately. This study's purpose was to compare physician and patient perceptions of the same telephone call episode. METHODS: Family practice residents (n = 19) recorded all patient-initiated after-hours telephone contacts (n = 192) during July 1993. Study personnel then telephoned, within 1 week of their call, the patients who made the calls. Patients were asked about the reason for their call, its seriousness, and their satisfaction with the handling of their problem. RESULTS: During the study month, 1.1 after-hours calls were received for every 10 office visits. A substantial minority of patients (29%) rated their problems in the highest severity category, while physicians assigned only 8% of calls the highest severity rating. The majority of patients (76.7%) were satisfied with how their after-hours calls were handled. CONCLUSIONS: In matched cases, physicians and patients perceive about the same proportion of calls to be routine versus more severe. Although patient satisfaction was high, further research into causes of dissatisfaction is needed.

Adult↗
PubMed1995

c-fos mRNA is present in axons of the hypothalamo-neurohypophysial system of the rat.

In situ hybridization revealed that c-fos encoding transcripts occur in the median eminence of rats. Osmotic stress resulted within 15 min. in additional labeling of the magnocellular nuclei, while osmotically stimulated rats that had been pretreated with colchicine failed to show c-fos hybridization in the magnocellular perikarya. Rats, pretreated with a polymerase II inhibitor, showed 15 min. after osmotic stimulation c-fos hybridization in the hypothalamic nuclei, whereas the median eminence was depleted of hybridization signal. c-fos is probably among the transcripts, stored in axons of the hypothalamo-neurohypophysial system, to be transported retrogradly upon osmotic stimulation.

Animals↗
PubMed1994

Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide induces anxiolytic effects in rat.

Antisense oligodeoxynucleotide complementary to the start coding region of rat corticotropin releasing hormone (CRH) mRNA was intracerebroventricularly infused into rats three times at 12-h intervals. In the shuttle-box avoidance procedure antisense-treated rats showed, within 6 h, significant acceleration and increase in the total number of discriminative avoidance responses compared with controls, treated with the corresponding sense probe or vehicle alone. Following the shuttle-box experiment hypothalamic CRH hybridization signals and immunoreactivity were reduced, while CRH immunoreactivity in the median eminence remained unchanged. Plasma ACTH and corticosterone were decreased in antisense-treated animals. It is likely that in addition to a selective blockade of CRH translation, antisense treatment may also interrupt secretion of CRH. Antisense targeting of the hypothalamo-hypophysial-adrenal axis may provide new strategies for the neuropharmacology of affective disorders.

Animals↗
PubMed1994

Ventral tegmental area (VTA) injections of tyrosine hydroxylase phosphorothioate antisense oligonucleotide suppress operant behavior in rats.

Dopaminergic A 10 neurons are known to be the essential part of the brain reinforcement system. An antisense oligonucleotide corresponding to the start coding region of rat tyrosine hydroxylase (TH) mRNA, the transcriptional message of the rate limiting enzyme in the metabolic pathway leading to catecholamine synthesis, was constructed and injected into the ventral tegmental area (VTA). 36 h after injection operant behavior was markedly reduced, and this suppression was fully reversed within 5 days following the antisense injection. Accordingly, TH immunoreactivity in the VTA was reduced in comparison to control experiments using mixed bases oligonucleotides. Our results demonstrate that highly specific inhibition of TH expression can be accomplished in the intact mesolimbic system by antisense treatment, thus providing a novel tool for studies on motivational processes in vivo.

Animals↗
PubMed1994

Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide treatment attenuates social defeat-induced anxiety in rats.

1. The neuropeptide corticotropin-releasing hormone (CRH) is the main mediator of the neuroendocrine and behavioral response to stress. End-capped phosphorothioate antisense and sense oligodeoxynucleotides (ODN) corresponding to the start coding region of rat CRH mRNA were infused intracerebroventricularly (30 micrograms/3 microliters per injection) three times at 12 hr intervals. Six hours after the last injection rats were subjected to social defeat stress and subsequently tested on the elevated plus maze. 2. Socially defeated CRH antisense-treated rats displayed markedly reduced anxiety-related behavior, as they spent significantly more time in the open arms of the plus maze compared to sense ODN- and vehicle-treated animals. 3. In controls, social defeat evoked a stress-induced elevation of CRH mRNA and CRH in the hypothalamus and a significant increase in plasma corticotropin (ACTH) levels. These parameters were attenuated in antisense-injected rats. 4. Our results suggest that CRH antisense treatment is effectively suppressing the neuroendocrine and behavioral effects of social defeat.

Agonistic Behavior↗
PubMed1994

Localization of tyrosine hydroxylase mRNA in the axons of the hypothalamo-neurohypophysial system.

With in situ hybridization we examined the localization of mRNA coding for tyrosine hydroxylase (TH) in the rat hypothalamo-neurohypophysial system (HNS) under conditions of acute osmotic stress. Fifteen min after salt loading, hybridization signal of TH mRNA could be located in the magnocellular hypothalamic nuclei and in the median eminence (ME). In untreated animals, TH mRNA was detected only in the ME. In osmotically challenged animals that had been pretreated with colchicine, signals for TH mRNA remained confined to the ME, while pretreatment of salt loaded rats with a polymerase II transcription inhibitor resulted in labelling of the magnocellular perikarya but a decrease of the hybridization signal in the ME. Our results suggest that also TH mRNA is among the RNAs which are axonally transported in the HNS. TH mRNA can probably be stored in axons of the hypothalamo-neurohypophysial tract, to be transported retrogradely and translated upon certain stimuli.

Animals↗
PubMed1994

Antisense oligodeoxynucleotides for in vivo targeting of corticotropin-releasing hormone mRNA: comparison of phosphorothioate and 3'-inverted probe performance.

Antisense DNA has been successfully used in vivo to selectively inhibit expression of proteins in the brain. However, stressful side effects after oligodeoxynucleotide (ODN) application have been observed, but not carefully characterized. An attempt was made to establish an animal model of reduced corticotropin-releasing hormone (CRH) activity, using antisense DNA corresponding to the start coding region of rat CRH mRNA with either 3'-3' inverted internucleotidic linkage or with all-phosphorothioate modification. Probes were injected intracerebroventricularly (i.c.v.) twice, 12-hours apart. After phosphorothioate sense ODN injection serum corticosterone levels were significantly elevated compared to vehicle (aCSF) or 3'-3' end inverted sense ODN controls. This increase was also apparent but less pronounced in phosphorothioate antisense treated animals compared with the corresponding sense group. After exposure to ether vapour, both phosphorothioate and inverted antisense ODN injected rats showed a markedly diminished stress induced corticosterone secretion compared to the corresponding sense or vehicle injected rats. These results indicate that a) stress induced corticosterone release is suppressed by i.c.v. CRH antisense treatment, b) phosphorothioate ODNs exert an unspecific, chronic stress-like activation of the HPA-axis and c) this effect is partly inhibited by phosphorothioate antisense directed against CRH mRNA.

Animals↗
PubMed1994

Vasopressin antisense oligonucleotide induces temporary diabetes insipidus in rats.

The purpose of this study was to downregulate the transcriptional message of arginine vasopressin (AVP) by antisense treatment. A complete phosphorothioate antisense oligodesoxynucleotide corresponding to the beginning of the coding region of rat AVP mRNA was constructed and injected into the lateral ventricle of rats. Within 3-6 h animals exhibited a temporary diabetes insipidus, which lasted up to 9 h. Accordingly, vasopressin immunoreactivity in the hypothalamic nuclei was reduced. Our results demonstrate that a specific and reversible inhibition of neuropeptide expression can be accomplished in the intact hypothalamo-neurohypophysial system by antisense treatment, thus providing a novel tool for studies on stimulus-secretion coupling in vivo.

Animals↗
PubMed1994

Molecular anatomy of a skin gland: histochemical and biochemical investigations on the mucous glands of Xenopus laevis.

The purpose of this study was to investigate the structure and chemical composition of the mucous skin gland of Xenopus laevis by combined morphological and biochemical techniques. Protein backbones of mucins were localized immunohistochemically in the gland with anti-peptide antibodies. Acid mucins were demonstrated by conventional histochemical techniques and their terminal carbohydrate residues were localized by lectin histochemistry. A close correlation between antibody and lectin binding of the same glycoproteins was achieved on Western blots from isolated skin gland mucins, indicating that the lectin-binding sites were due to defined mucin molecules. The cone cell, thought to be a degenerative cell in the past, contained mucin granules with an electron-dense core, strong PAS reactivity, a special lectin-binding pattern, and localization of integumentary mucins FIM-B.1 and FIM-C.1. These results indicate that cone cells are a distinct cell type, elaborating and releasing particular mucins, and that functional heterogeneity of mucus-producing cells exists in the mucous skin glands of X. laevis.

Animals↗