Biomedical subjects

J A Hirsch

Publications and source records attributed to J A Hirsch.

At least 19 recordsLinked to original sources

PubMed1997

Structure of the multimodular endonuclease FokI bound to DNA.

FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein-protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.

Amino Acid Sequence↗

PubMed1997

Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.

FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 A in resolution and belong to space group P2(1) with unit cell dimensions of a=67.9 A, b=119.8 A, c=69.1 A, beta = 96.6 degrees. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease.

Crystallography, X-Ray↗

PubMed1997

Association between blood pressure and lifetime drinking patterns in moderate drinkers.

OBJECTIVE: To compare the relationship between blood pressure and recent vs lifetime alcohol intake measures in a nonalcoholic cohort using measures that normalized alcohol intake in terms of the volume of distribution for ethanol (total body water) for each subject. METHOD: Correlations between blood pressure and alcohol intake measures were determined in a cohort of 84 (48 male) normotensive moderate drinkers, using multiple linear regressions to correct for the influence of potentially confounding variables. RESULTS: Significant correlations were observed between systolic pressure and measures of both recent and lifetime alcohol consumption in men. Diastolic pressure was found to be significantly associated only with the duration of lifetime drinking, and only in women. Measures of lifetime total dose were not significantly correlated with either systolic or diastolic pressure. Alcohol intake expressed in terms of the number of "drinks" per drinking day was as highly associated with blood pressure (systolic) in men as were measures that normalized alcohol intake in terms of the body water content of each subject. A measure of the mean number of drinks consumed on drinking occasions over the drinking career produced the highest correlations with systolic pressure. CONCLUSIONS: Measures of both recent and lifetime alcohol intake may be relevant to the understanding of alcohol-blood pressure relationships.

Adult↗

PubMed1996

Residual pressor effects of chronic alcohol in detoxified alcoholics.

Although research in population studies has indicated that recent alcohol intake is positively correlated with blood pressure, there is a need to study the relationship of blood pressure to measures of lifetime alcohol intake in alcoholics. To this end, we assessed systolic and diastolic pressures and lifetime alcohol intake through structured interviews with 253 normotensive recovering alcoholics. Blood pressures were first corrected with multiple linear regression for the influence of confounding or modifying variables and then were regressed against alcohol consumption measures. Systolic pressure was significantly correlated (positively) with only a few measures of recent alcohol intake, and the correlations were not high (r2 = .05 to .11, P < .05). Diastolic pressure was found to be highly and positively correlated with the duration of the drinking career, but more so in blacks than in whites. The total lifetime dose of alcohol was found to be positively correlated with diastolic but not systolic pressure, but only in black male alcoholics. The steeper slope of the regression of blood pressure versus lifetime total alcohol or duration of the drinking career in black alcoholics suggests greater cardiovascular susceptibility to alcohol toxicity as lifetime doses increase and as the drinking career lengthens.

Adolescent↗

PubMed1996

Clinical assessment of MR of the brain in nonsurgical inpatients.

PURPOSE: To evaluate the effect of MR imaging of the brain on four domains of patient care: diagnosis, diagnostic workup, therapy, and prognosis. METHODS: Pre- and post-MR written questionnaires and oral interviews were administered to the referring clinicians of 103 medical and neurologic inpatients at a tertiary care institution. Additional information was obtained from radiologic reports and records. RESULTS: The study population had a diverse array of signs and symptoms and of presumptive clinical diagnoses, reflecting the breadth of disease seen at our institution. The vast majority of physicians (89%) reported that MR imaging added significant diagnostic information, playing an important role in guiding diagnostic workup (24%), planning treatment (34%), and estimating prognosis (47%). MR imaging was significantly more likely to decrease than to increase confidence in the presumptive clinical diagnosis. Thus, MR imaging may be most useful in the setting of diagnostic uncertainty. CONCLUSION: Our results show that MR imaging of the brain has important effects on each of the four domains of care for medical inpatients.

Brain↗

PubMed1995

Structure of the even-skipped homeodomain complexed to AT-rich DNA: new perspectives on homeodomain specificity.

even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly. Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich, the other GC-rich. We describe a crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution. The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion. This arrangement suggests a mechanism for the homeoproteins' regulatory specificity. In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases.

Amino Acid Sequence↗

PubMed1995

Visually evoked calcium action potentials in cat striate cortex.

Early intracellular studies of cerebral cortical neurons indicated that synaptic input evokes dendritic action potentials that convey information towards the soma. Subsequent work in vitro established that neocortical neurons produce dendritic Ca2+ action potentials. To determine whether natural stimuli elicit Ca2+ spikes, we combined the techniques of whole-cell recording, pharmacology and quantitative receptive field mapping. Our findings show that visual stimulation routinely evoked Ca2+ spikes in distinct functional and anatomical classes of cells in different layers of the cat striate cortex. Hence regenerative Ca2+ potentials appear to play a role in both the initial and later stages of cortical sensory processing.

Action Potentials↗

PubMed1995

Synaptic integration in layer IV of the ferret striate cortex.

1. Whole-cell patch recording were made with dye-filled electrodes from layer IV in slices of the ferret striate cortex. Projections from the thalamus and layer VI provide most of the extralaminar input to layer IV. Interactions between these two pathways are thought to play a role in the generation of suppressive non-linearities such as end-inhibition. Thus, synaptic responses evoked by stimulating each pathway individually were compared with those produced by activating both projections together. 2. Spiny stellate cells are the majority population in layer IV and were the most frequently patched neurons (n = 23); all fired adapting trains of large, fast action potentials. About half of those tested (n = 13) were progressively inhibited by strengthening the stimulus to layer VI, while the rest became more excited. For the former, the response evoked by stimulating both pathways in coincidence was often more hyperpolarizing than would have been predicted by summing the responses to either projection alone (n = 4). Hence, the inputs from the thalamus and layer VI are integrated by circuits that can produce strong and non-linear inhibition. 3. Recordings from various basket cells, which are inhibitory, have provided a first view of the suppressive circuits in layer IV (n = 5). Two cells were excited by stimulation of both pathways. The remaining three cells were mainly excited by activation of thalamic afferents but were largely inhibited by stimulation of layer VI. Thus, inhibition seen at the level of the spiny stellate cells could result from two mechanisms operating via presynaptic smooth cells: convergent excitation provided by both ascending pathways on the one hand, and a push-pull relationship between pathways on the other.

Animals↗

PubMed1995

Purification, crystallization, and preliminary X-ray diffraction analysis of even-skipped homeodomain complexed to DNA.

Embryonic development in metazoa, to a significant extent, is directed by genes which contain a conserved sequence motif named the homeobox. This sequence encodes a polypeptide called the homeodomain which has sequence specific DNA-binding activity. We report the purification, crystallization, and preliminary diffraction analysis of the Drosophila Even-skipped homeodomain (Eve HD) bound to two different oligonucleotides. Crystals of Eve HD complexed with an AT-rich sequence belong to space group P2(1), a = 34.06, b = 61.61, c = 39.99 Angstrom, beta = 90.0 degrees. These crystals diffract to at least 2.0 Angstrom and both native and derivative data sets have been collected. Crystals of Eve HD complexed with a GC-rich sequence belong to space group P6(3), a = b = 124.52, c = 66.78 Angstrom and diffract to 3.5 Angstrom resolution. A native data set has been collected.

Amino Acid Sequence↗

PubMed1995

Depression of an inhibitory reflex, the masseteric silent period, in recovering alcoholics.

Because inhibitory deficits may contribute to motor incoordination of alcoholics, we proposed that the duration of the masseteric silent period (SP), an inhibitory reflex, might be shorter in alcoholics (ALs) than in nonalcoholics (NAs). To test this hypothesis, we measured the SP in a racially mixed group of 12 ALs and 12 NAs matched for age (31-49 years) and sex. All subjects were normotensive, had full dentition, and reported no major medical problems. Sensory and motor conduction velocities of the anterior tibialis, ulnar, and medial nerves were measured for each subject, and were not different in ALs and NAs. Jaw jerk and SP were evoked by tapping the chin manually with a rubber hammer containing a circuit to trigger the oscilloscope display of the masseter EMGs. Averaged mean latencies and durations of the SPs were not significantly different between the right and left masseters. SP latencies were significantly (0.7 msec) shorter in ALs than NAs (p < 0.01). The mean SP duration for ALs (13.9 +/- 1.6 msec) was also significantly shorter than that of the NAs (24.4 +/- 2.4 msec; p < 0.001). Results support the hypothesis that chronic alcoholism may interfere with inhibitory mechanisms in the CNS.

Adult↗

PubMed1995

Drinking patterns and health status in smoking and nonsmoking alcoholics.

Alcoholics who smoked also reported that they drank more frequently and consumed more alcohol on drinking occasions than alcoholics who did not smoke, a practice that resulted in a substantially greater lifetime alcohol consumption in the smokers. Smoking alcoholics also consumed more cigarettes and reported more smoking-related physical symptoms than social drinkers who smoked. The heart rates (HRs) of smoking and nonsmoking alcoholics were similar and both exceeded the HRs for the smoking social drinkers by approximately 13 beats/min (bpm) in males and by approximately 7 bpm in females. Surprisingly, correlations between HR and lifetime alcohol consumption were higher and slopes were steeper in controls than in alcoholics. HRs in a subset of the male alcoholics fell only approximately 3 bpm after 24 weeks of abstinence, but changed no further over an additional 24-week period. Taken together, the findings suggest that HRs may have been higher in this group of alcoholics before the onset of alcohol abuse and that alcohol intake contributed only slightly to the high HR.

Adult↗

PubMed1995

Small-cell carcinoma of the esophagus with regression after combination chemotherapy and radiation therapy.

The authors present an unusual case of small-cell carcinoma of the esophagus, which manifested on double-contrast esophagography as an ulcerated submucosal mass. The lesion underwent dramatic regression after combination chemotherapy and radiation therapy, which has occasionally been used as an alternative to surgery in patients with this rare but aggressive esophageal neoplasm.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1994

Control of breathing patterns and abdominal muscles during graded loads and tilt.

Tilting from supine to upright purportedly enhances both segmental and pulmonary proprioceptive feedback, whereas an expiratory threshold load (ETL) preferentially enhances pulmonary feedback. To test this we studied 13 adults when supine and 60 degrees and 90 degrees head up. We measured tidal volume, inspiratory duration (TI), and expiratory duration (TE) from flow; estimated end-expiratory lung volume from inspiratory capacity; and determined burst amplitudes and durations from abdominal electromyograms (EMGs). ETLs were incremented from 0 (control) to 25 cmH2O in 5-cmH2O steps. Tidal volume was significantly increased by ETL but was unaffected by body position. Every load prolonged TE, whereas TI remained unchanged. When subjects were supine, abdominal EMGs were silent but became tonically active when subjects were upright. During ETL, abdominal activity became rhythmical and phase locked to expiration. Bursts amplitudes were enhanced with each increment in ETL, but burst durations did not change even though TE was prolonged. The altered breathing pattern and active expiration augmented inspiratory flow and decreased end-tidal PCO2. Responses were greatest when subjects were 90 degrees head up. The load-related increments in abdominal recruitment, with no change in burst durations, fit the concept of two central pattern generators: one controlling pattern and the other controlling rhythm of the central respiratory drive.

Abdominal Muscles↗

PubMed1993

Long-term changes in synaptic strength along specific intrinsic pathways in the cat visual cortex.

1. The dense system of horizontal connections that arise and course within the striate cortex are thought to inform single cells about stimuli arising in disparate points in visual space and to modulate responses evoked from within the receptive field. To learn whether or not the strength of the horizontal connections could vary over the long term, and if such changes could affect the integration of vertical, interlaminar inputs, we have recorded intracellularly from the superficial layers in slices of the adult cat's visual cortex. 2. The monosynaptic EPSP evoked by stimulating horizontal fibres showed long-term facilitation in twelve of the twenty cells that were conditioned by repetitively pairing synaptic responses with depolarizing pulses of current; the maximum increase observed was 200%. Strong inhibition present in the postsynaptic response usually indicated that facilitation would not occur. 3. In instances where horizontal input evoked both mono- and polysynaptic EPSPs, both early and late events showed facilitation, with the most dramatic enhancement contributed by the polysynaptic components. 4. For the twenty-eight cells whose responses to stimulation of interlaminar as well as horizontal pathways were assessed, all were found to receive non-overlapping inputs from each source. Conditioning produced long-term changes in the strength of the interlaminar inputs. 5. Changes in synaptic strength were usually confined to the conditioned pathway, though in four out of twenty-six times we observed heterosynaptic facilitation of polysynaptic EPSPs. 6. The conditioning protocol led to lasting depression rather than facilitation in three out of eleven instances; the reduction was only observed in the multisynaptic components. 7. We suggest that the synaptic changes observed here may be related to certain dynamic changes in receptive field properties that have been characterized in vivo.

Animals↗

PubMed1993

Recovery of respiratory sinus arrhythmia in detoxified alcoholic subjects.

Although most alcoholic subjects show little autonomic dysfunction, severe alcoholic subjects may have pathological changes in autonomic nerves. We asked if respiratory sinus arrhythmia amplitude (RSA), an index of vagal cardiac control, is decreased in alcoholism and, if so, whether the decrease is reversed with abstinence. RSA was assessed in 17 normotensive alcoholic subjects (A) at 1, 4, 12, and 24 wk of abstinence after detoxification and at similar intervals in 17 controls (C) matched for age, race, and gender. Subjects were studied in both supine and seated positions while breathing in a prescribed deep (> 50% vital capacity) and slow (5-7/min) pattern. Mean heart rate (HR) was determined over 30 s from the electrocardiogram; RSA (the difference between maximum and minimum instantaneous HRs after inspiratory onset) was determined from 10 consecutive breaths. In C, both HR (supine: 61.5 +/- 2.2 beats/min; seated: 71.3 +/- 1.7 beats/min; P < 0.002) and RSA (supine: 22.5 +/- 1.0 beats/min; seated: 28.4 +/- 1.4 beats/min; P < 0.003) were higher when seated than when supine, but neither HR nor RSA varied over 24 wk. At week 1 of abstinence, HRs for A were higher than those for C (supine: 74.2 +/- 2.3 beats/min, P < 0.001; seated: 83.2 +/- 2.7 beats/min, P < 0.003), but by week 24, both seated and supine values returned to control levels. RSA in A at week 1, was only one-half that of C (supine: 11.1 +/- 1.4 beats/min, P < 0.001; seated: 14.7 +/- 1.9 beats/min, P < 0.001) and independent of body position.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Respiratory and abdominal muscle responses to expiratory threshold loading in cystic fibrosis.

We hypothesized that the hyperinflation and pulmonary dysfunction of cystic fibrosis (CF) would distort feedback and therefore alter the abdominal muscle response to graded expiratory threshold loads (ETLs). We compared the respiratory and abdominal muscle responses with graded ETLs of seven CF patients with severe lung dysfunction with those of matched healthy control subjects in the supine and 60 degrees head-up positions. Breathing frequency, tidal volume, and ventilatory timing were determined from inspiratory flow recordings. Abdominal electromyograms (EMGs) were detected with surface electrodes placed unilaterally over the external and internal oblique and the rectus abdominis muscles. Thresholds, times of onset, and durations of phasic abdominal activity were determined from raw EMGs; peak amplitudes were determined from integrated EMGs. Graded ETLs were imposed by submerging a tube from the expiratory port of the breathing valve into a column of water at depths of 0-25 cmH2O. We found that breathing frequency, tidal volume, and expired minute ventilation were higher in CF patients than in control subjects during low ETLs; a change in body position did not alter these ventilatory responses in the CF patients but did in the control subjects. All CF patients, but none of the control subjects, had tonic abdominal activity while supine. CF patients recruited abdominal muscles at lower loads, earlier in the respiratory cycle, and to a higher recruitment level in both positions than the control subjects, but burst duration of phasic activity was not different between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Abdominal Muscles↗

PubMed1991

Synaptic physiology of horizontal connections in the cat's visual cortex.

Horizontal connections are a principal component of intrinsic cortical circuitry. They arise mainly from pyramidal cells and course parallel to the brain's surface for distances as long as 8 mm, linking columns with shared orientation preference and allowing cells to integrate visual information from outside their receptive fields. We examined the synaptic physiology of the horizontal pathway in slices of the cat's striate cortex and found that activating lateral fibers produced both excitation and inhibition. We recorded the postsynaptic responses of identified pyramidal cells in layer 2 + 3 of area 17 to electrical shocks applied at three sites: in the home column of the impaled neuron either in layer 2 + 3 or 4, or at a lateral distance of 0.9-3 mm in layer 2 + 3. Within the home column, suprathreshold stimuli produced compound EPSPs with action potentials, followed by fast, GABAAergic IPSPs and a slower, GABABergic IPSP. For the distant stimulating site, the threshold response was an EPSP. Stronger shocks frequently evoked a disynaptic, GABAAergic IPSP that truncated the EPSP and could dominate the postsynaptic response. At the resting potential, the horizontally evoked EPSP was too small to elicit spikes. With depolarization of the membrane, however, it grew several hundred-fold. This amplification was blocked by N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), but not by 2-amino-5-phosphonovalerate (APV), indicating that it was mediated by Na+ channels, rather than by NMDA receptors. We propose that the horizontal connections provide the means for stimuli outside the receptive field to modulate activity elicited within its confines. The voltage-dependent enhancement of the laterally evoked EPSP may explain why stimulating the surround by itself fails to drive cells but can facilitate their response to stimuli within the receptive field. The ability to initiate disynaptic inhibition from lateral sites shows that recruiting appropriate groups of horizontal fibers can also have a suppressive effect. Thus, the effect of horizontal input is state dependent, with the size and sign of the laterally evoked response changing according to the balance of converging inputs.

2-Amino-5-phosphonovalerate↗

PubMed1990

Origin and selection of peripheral CD4-CD8- T cells bearing alpha/beta T cell antigen receptors in autoimmune gld mice.

We have analyzed the origin and development of unusual CD4-CD8- alpha/beta T cell receptor-positive peripheral T cells produced in large numbers by mice homozygous for the gld mutation (C3H-gld/gld). These mice may be an important model for investigating processes controlling T cell development. Bone marrow transfers demonstrated that the gld defect was intrinsic to bone marrow-derived cells. Clonal deletion of potentially autoreactive cells was observed in peripheral gld CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells, as well as mature thymocytes. This suggests that gld CD4-CD8- T cells have passed through the thymus in ontogeny and that gld autoimmunity does not result from a general defect in elimination of self-reactive thymocytes. These observations, combined with demethylation of the CD8 gene in the CD4-CD8- population, support prior expression of CD4 and/or CD8 in gld CD4-CD8- T cell ontogeny, perhaps at a CD4+CD8+ stage. Steroid sensitivity of gld thymocytes and CD4-CD8- T cells was normal. Therefore, we found no gross abnormalities in two major mechanisms of inducible cell death in the gld thymus, the clonal deletion process associated with tolerance and the steroid-inducible endogenous endonuclease thought to be involved in apoptosis of unselected thymocytes. The data suggest that if gld CD4-CD8- T cells arise via escape from normal elimination in the thymus, they must do so by a novel defect in thymic selection (perhaps related to aberrant positive signals) and/or are expanded by an extrathymic process which allows clonal deletion to occur.

Animals↗