Systemic tocainide relieves mechanical hypersensitivity and normalizes the responses of hyperexcitable dorsal horn wide-dynamic-range neurons after transient spinal cord ischemia in rats.

In the present study we examined the effect of systemic tocainide on sensory hypersensitivity in rats after spinal cord ischemia induced by a photochemical technique. After induction of spinal cord ischemia the rats exhibited a sensory disturbance which was mainly expressed as vocalization to innocuous cutaneous mechanical stimuli (allodynia) in the flank area during the following several days. Tocainide at 75 mg/kg i.p., but not 50 mg/kg i.p., significantly increased the vocalization threshold to mechanical pressure for 2 h. The effect of intraarterial (i.a.) tocainide on the responses of dorsal horn wide-dynamic-range (WDR) neurons to suprathreshold electrical stimulation of their receptive fields was also examined in normal rats and after transient spinal cord ischemia, at a time when the animals exhibited typical behavioral allodynia in the dermatomes innervated by the ischemic spinal segments. In normal rats, tocainide (50 mg/kg i.a.) strongly suppressed the responses of WDR neurons to C fiber input with lesser effect on A fiber input. In allodynic rats, tocainide suppressed the augmented A and C fiber mediated responses of WDR neurons to the extent that their responses were similar to those seen in normal rats without tocainide. There was no difference in the overall depression of A and C fiber mediated input by tocainide between normal and allodynic rats. The present results demonstrated the analgesic effect of systemic tocainide in relieving allodynia in rats and indicated that systemic local anesthetics, at doses that do not block nerve conduction, can be effective in suppressing dorsal horn WDR neuronal activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Low-threshold, short-latency cutaneous reflexes during fictive locomotion in the "semi-chronic" spinal cat.

Low-threshold, short-latency cutaneous reflexes evoked in ipsilateral hindlimb motor nerves were examined during fictive locomotion. Locomotion in 11 anaemically decerebrated spinal animals (1-3 weeks after transection at T13-L1) was induced by administration of clonidine, L-dopa and nialamide; by administration of the latter two drugs only; or by exteroceptive stimulation in the absence of any drugs. The caudal and lateral cutaneous sural, caudal cutaneous femoral, saphenous and superficial peroneal nerves were stimulated at low threshold (1.5-3 T). Pooled results from all combinations of cutaneous nerves stimulated and muscle nerves recorded show that the initial response was excitatory in 40 of 50 triceps surae and 17 of 20 semitendinosus (St) electroneurograms (ENGs). These excitatory responses occurred at latencies that ranged from 5 to 15 ms and tended to be maximal during the motor nerve's active period in the step cycle (i.e. they were modulated in a phase-dependent manner). Only three inhibitory responses (9-12 ms earliest latency) were encountered in total: in two St ENGs of one animal and in one lateral gastrocnemius-soleus ENG of a different animal. In two animals a "second" excitatory response (15-25 ms latency) was sometimes recorded in triceps surae and St nerves and, interestingly, could be modulated out of phase with the early response. Weak short-latency excitatory reflexes were also found in contralateral St ENGs when examined. Finally, among medial gastrocnemius, lateral gastrocnemius and soleus nerves, excitatory responses due to stimulation of any particular cutaneous nerve tended to be modulated similarly but were of consistently different amplitude among the three. This finding, together with the general observation that excitatory reflexes produced by stimulation of a particular cutaneous nerve were modulated similarly in extensors (or flexors) of different animals, suggests that spinal circuits generating locomotion may indeed exert a stereotypic control over interneurons in specific cutaneous reflex pathways to motoneurons. The results are primarily discussed in terms of the existing evidence for short-latency excitatory cutaneous reflexes in extensors in a variety of locomotive and non-locomotive preparations.


Observations on the development of transplanted embryonic ventral horn neurones grafted into adult rat spinal cord and connected to skeletal muscle implants via a peripheral nerve.

Embryonic spinal cord grafts from 12-day-old rat embryos were placed into the lumbar spinal cord of adult rats depleted of sciatic motoneurones by a neonatal nerve injury. A soleus muscle was removed from the leg and implanted paravertebrally, the proximal end of its nerve connected to the graft site. Fluorescent retrograde tracers injected into the soleus implant, 37-64 days postoperatively, labelled neurones that had grown axons to the muscle. Approximately one-fifth of retrogradely labelled neurons were within the graft; however, the majority were found within the host spinal cord close to the graft. These included large neurons within the motoneurone-depleted dorsolateral ventral horn. In control experiments a muscle and nerve were implanted but no embryonic tissue grafted. Significantly fewer neurones were labelled. In some animals, one tracer was injected into the soleus muscle whilst another was applied to the cut sciatic nerve ipsilateral to the graft site. No neurones were found to project axons to both targets. In animals that received grafts prelabelled with bromodeoxyuridine (BrDU) some neurones were found to be both BrDU positive and retrogradely labelled from the soleus implant. These were most frequently within the motoneuron-depleted ventral horn ipsilateral to the graft. Thus, grafted neurones may migrate to an appropriate location within the host neuropil. Acetylcholinesterase (AChE) histochemistry showed the graft site contained immature but AChE-positive neurones. Some regions of host ventral horn contained unusually few AChE-positive nerve fibres and occasional large AChE-positive neurones, some of which were also retrogradely labelled from the implanted muscle. Studies of implanted soleus 21-90 days after transplantation showed that muscle fibres, after initial degeneration, regenerated displaying differing phenotypes, presumably under the influence of new motor innervation.


Responses of barrel cortex neurons in awake rats and effects of urethane anesthesia.

A "barrel" is an interconnected network of layer IV neurons that is an important component of a functional cortical column in the whisker area of the rodent primary somatosensory cortex. The present study was undertaken in order to resolve apparently conflicting findings from single-unit studies of barrel neurons conducted in rats maintained under different anesthetic conditions. Multiunit responses to controlled deflections of mystacial vibrissae were recorded from the whisker/barrel cortex of awake, undrugged rats, and responses at the same recording site were reexamined after the animal was anesthetized with urethane. In contrast to the awake condition, stimulus-evoked responses under urethane were characterized by a large late component. Such effects were more pronounced for deflections of noncolumnar or "adjacent" whiskers than for the columnar whisker. Latencies to peak responses were virtually identical for the columnar whisker in awake and urethane states (11.9 vs 11.8 ms) but were considerably longer for adjacent whisker deflections in urethane-anesthetized animals (15.5 vs 29.0 ms). The magnitudes of adjacent whisker responses, relative to the response evoked by the columnar whisker, varied with the laminar location of the recording site in awake but not in urethane-anesthetized animals; in awake rats, receptive fields were clearly smallest in the layer IV barrels. Results in the awake condition confirm those of previous studies conducted in unanesthetized or lightly sedated animals, and data obtained with urethane are comparable to others' results in urethane-anesthetized rats. The former have important implications for how barrel cortex processes information in behaving animals.


Pretectal jerk neuron activity during saccadic eye movements and visual stimulations in the cat.

The activity of 'jerk neurons' was recorded extracellularly in the pretectum of the awake cat. The characteristic response of jerk neurons was a short, high-frequency burst that occurred after fast movements ('jerks') of a large, structured visual stimulus, during saccadic eye movements in the light, and after 'on' or 'off' visual stimulation. Mean burst latency to pure visual 'jerks' was 50 ms, whereas it was 30 ms to saccadic eye movements. Bursts were found to be stereotyped; the highest discharge rate was always at burst onset. Jerk neurons were not selective for stimulus parameters (such as movement amplitude or direction) except that in some neurons a weak correlation between stimulus velocity and discharge frequency was found. During saccades in the dark, clear bursts were only rarely found. In about half of the neurons, however, there was a slight but significant increase in the number of spikes above spontaneous frequency. Visual receptive fields were very large (46 degrees horizontal and 35 degrees vertical extent, on average). Nevertheless, the pretectal jerk neurons showed a rough retinotopic order, which was in accordance with the published retinotopy of the pretectum. Jerk neurons were found throughout the whole superficial pretectum, but preferentially in an area that corresponds to the nucleus of the optic tract (NOT) and the nucleus pretectalis posterior (NPP). Saccades were elicited by electrical stimulations at the sites where jerk neurons were recorded. The direction of the elicited saccades depended strongly on the pretectal stimulation site. A possible role of the jerk neurons as a visuomotor relay to elicit saccades or to modulate perception and attention is discussed.


Transcallosally evoked responses in the visual cortex of normal and monocularly enucleated rabbits.

In visual cortex of normal adult rabbits, callosal projections are restricted to a 2 mm wide band at the area 17/18 border. In adult rabbits which are monocularly enucleated (ME) on the day of birth, the callosal zone extends 4 mm into the medial region of area 17 in the cortex ipsilateral to the remaining eye. In this study, the function of these anomalous callosal projections in ME rabbits was investigated using electrophysiological techniques. A microelectrode was placed in the visual cortex ipsilateral to the enucleated eye at the 17/18 border, bipolar stimulating electrodes were placed in a homotopic location in the contralateral cortex, and averaged evoked responses (AERs) to stimulation were recorded. The stimulating electrodes were then moved mediolaterally in 1 mm steps, and the AERs were recorded for each location of the stimulating electrodes. In the normal rabbit, a maximal short latency evoked response was recorded when the stimulating electrodes were at a location homotopic to the recording electrode. When the stimulating electrodes were moved a distance of 1 mm or more from this optimal position, this short latency response was either absent or dramatically decreased in amplitude, reflecting the precise topographic pattern of the normal callosal projection. In contrast, in ME rabbits, a consistent response was evoked at the 17/18 border when the stimulating electrodes were moved as much as 3 mm medial to the homotopic position. Since antidromically activated responses and both pre- and postsynaptic orthodromically activated responses contribute to the AER, recordings were also made from single cells in some animals.(ABSTRACT TRUNCATED AT 250 WORDS)


The effects of monocular enucleation on visual topography in area 17 in the rabbit.

The effects of neonatal monocular enucleation on the topographic representation of the ipsilateral visual field in the visual cortex of the rabbit were investigated, using electrophysiological recordings of multi-unit activity in area 17. Topography of receptive fields was determined in normal adult rabbits, adult rabbits monocularly enucleated on the day of birth and adult rabbits monocularly enucleated as adults. In normal rabbits and in adult enucleates, the projection from the ipsilateral eye is represented by a strip of cortex extending approximately 2 mm from the 17/18 border. In neonatal enucleates, the width of the area of cortex in which the projection from the ipsilateral eye is represented was approximately twice as large as normal. Visual topography was normal in the superior-inferior axis but was distorted in the nasotemporal axis. Our data suggest that the abnormal topography observed in the visual cortex of neonatally enucleated rabbits may play a major role in shaping the abnormal visual callosal projections observed in these animals. In addition, our data indicate that, following neonatal monocular enucleation, developmental abnormalities in the topography of geniculocortical projections can occur independently of any alteration in the retinogeniculate projection patterns.


Pattern of projections of group I afferents from elbow muscles to motoneurones supplying wrist muscles in man.

The pattern of projections of low threshold afferents from triceps and biceps brachii muscles onto motoneurones innervating muscles acting at the wrist was assessed by a reflex and a poststimulus time histogram (psth) technique. Activation of low-threshold afferents originating from elbow flexors or extensors resulted in an early, short-lasting inhibition of wrist flexor motoneurones (flexor carpi radialis, flexor carpi ulnaris). An inhibition was also found in the extensor carpi radialis (ECR) motoneurones after stimulation of low-threshold afferents from triceps. Evidence is presented that Ia fibres contribute to these effects. The inhibitory effects were found in all subjects, but they were constant in only 57% of the reflex experimental sessions and in 25% of the explored motor units. Stimulation of biceps low-threshold afferents was always ineffective on ECR motoneurones. No early facilitation was ever seen in motor nuclei innervating wrist muscles following stimulation of low threshold afferents from biceps and triceps. The pattern of transjoint projections of group I afferents from proximal to distal muscles and from distal to proximal ones (Cavallari and Katz 1989) is discussed in relation to that described in the cat forelimb.


The transcortical nature of the late reflex responses in human small hand muscle to digital nerve stimulation.

The hypothesis that long-latency reflex activity in human small hand muscles in response to stimulation of digital nerves involves a transcortical pathway was tested by combining digital nerve stimulation and magnetic stimulation over the motor cortex in 12 studies on nine normal subjects. Postsynaptic events in human single first dorsal interosseous (FDI) motoneurones were derived from changes in the firing probability of voluntarily activated single motor units. Electromagnetic stimulation over the contralateral motor cortex resulted in a short-latency, brief facilitation of FDI motor units considered to be due to the activation of "fast" corticospinal neurones making monosynaptic projections to motoneurones. Stimulation of the digital nerves of the index finger produced a period of reduced firing probability (I1), a period of increased firing probability (E2) and a further period of reduced firing probability (I2) in FDI motor units. When the two stimuli were given separately and then together, timed so that the magnetic stimulus occurred at the predicted transit time of the E2 through the cortex, the facilitation of FDI motoneurones by the combined stimulation was often less than the algebraic sum of the facilitations from each stimulus alone. Thus, in contrast to the results of similar studies on the late response to muscle stretch, there is no confirmation that the E2 from digital nerve stimulation is due to a transcortical reflex.


Synergistic finger movements in a skilled motor task.

When skilled typists strike one key, typically all of the fingers of one hand are in motion simultaneously. We studied the extent to which the motion of the individual fingers was similar. Subjects were asked to type lists of words, each word designed so that only one key would be pressed by a finger of one hand, the remaining letters being typed with the other hand. Changes in the lengths (flexion-extension) and orientation (abduction-adduction) of each finger were measured and the similarity of the motion of pairs of fingers was assessed by computing correlation coefficients. For each pair of fingers, the correlation coefficients were broadly distributed, but in most instances the mean was significantly greater than zero. Adjacent fingers showed a higher degree of correlation than did non-adjacent fingers. When one of the fingers was actually used to press the key, the degree of correlation decreased substantially. The results demonstrate that in this skilled motor task, the fingers usually tend to be moved together, but they can be moved independently when the task so requires.


Bidirectional control of saccadic eye movements by the disconnected cerebral hemispheres.

The present investigation demonstrates that callosotomy patient J.W. can generate either leftward or rightward saccades in response to color cues presented unilaterally. When asked to name the colors, performance was at chance for left visual field presentations, demonstrating a disability in interhemispheric transfer of chromatic information. The successful control of saccadic direction based on discriminative color cues that appear confined to a single hemisphere may suggest a capacity for bidirectional control of saccadic eye movements in the disconnected cerebral hemispheres.


The vertex-positive scalp potential evoked by faces and by objects.

The influence of stimulus form on the scalp-recorded "vertex positive peak" (VPP) evoked by images of faces (Jeffreys 1989a) was studied in seven subjects. In separate experiments, we recorded the responses to 2D images of: (1) many different depictions of human faces; (2) the heads of several different species; (3) many familiar non-face objects; and (4) stimuli where the configuration of objects were modified to produce an "illusory" or "non-contextual" subjective impression of a face. The results showed that every facial representation, including the "illusory" stimuli, and most of the non-face objects, evoked a VPP of corresponding form and scalp distribution. The object-evoked VPPs, however, were always smaller and usually later than those evoked by the faces. VPPs of longer latency but often comparable amplitude were also recorded for impoverished compared to well-defined facial representations; and for most non-human compared to human faces. Very consistent responses were recorded to repeated presentations of the same stimulus for the same subject, but there was considerable variation in latency as well as amplitude (but not form) of the VPP evoked under identical experimental conditions for different subjects. These response properties of the VPP, suggest that its underlying physiological generators are sensitive to basic configural properties of the visual stimulus; and also the face- and object-related information are processed in the same brain area(s), although not necessarily by the same physiological mechanisms.


Evoked potential evidence for human brain mechanisms that respond to single, fixated faces.

The influence of visual fixation position and stimulus size on the scalp-recorded "vertex positive peak" (VPP) evoked by images of faces was studied in three subjects. Responses were recorded, in turn, for line-drawn, frontal-view faces of approximately 8, 4, 2, and 1 deg length, fixated at the centre (bridge of the nose), and at points 1, 2, 3, and 4 deg to the left and right, and above and below, centre. The results showed that central fixation produced VPPs of similar, maximal amplitude for all face sizes. By comparison, "on-face" eccentric viewing yielded attenuated and delayed responses, and the degree of response attenuation as a function of eccentricity was directly related to the face size, with similar amplitude responses being evoked for corresponding fixation locations on each face. Very small or no VPPs were recorded for most "off-face" fixations. Similar results were observed for profile faces, except that the maximal VPP was recorded for fixations near the eyes and not in the centre of the head, and almost identical VPPs were evoked by a centrally fixated face presented with and without an adjacent face or object. These response properties, which correspond to the subjective perception of the facial stimuli, suggest that the VPP reflects brain mechanisms optimized to respond to single, fixated faces, irrespective both of facial image size and of the presence of neighbouring figures.


Do G protein subunits associate via a three-stranded coiled coil?

We used a computer-based prediction algorithm to identify probable coiled-coil segments at the N-termini of G protein alpha, beta and gamma subunits. This result indicates that G protein trimers may form via a three-stranded coiled coil. Previous biochemical results had shown that the N-termini of alpha and beta are involved in subunit interactions. Here we present a structural model for the N-terminal domain of beta gamma and a hypothesis for the reversible association of alpha to beta gamma.


Site-directed mutation makes rabbit calcyclin dimer.

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcylin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.


Amiloride at pH 7.0 inhibits the Na(+)-driven flagellar motors of Vibrio alginolyticus but allows cell growth.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na(+)-driven flagellar motors of Vibrio.


Determination of proteinase 3-alpha 1-antitrypsin complexes in inflammatory fluids.

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.


Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen.

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS-PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA-immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA, these CGA-processing products are released from the sheep spleen. The secretion of CGA-derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.


Detection of an enzyme bound gamma-glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli.

E. coli carbamyl phosphate synthetase binds 0.2-0.4 mol equivalents of glutamine in an acid resistant form. The bound material is quantitatively released as glutamate by weak base hydrolysis and as a mixture of 12% glutamate, 10% gamma-glutamylhydroxamate, and 70% pyrrollidonecarboxylic acid by hydrolysis with hydroxylamine. These results provide direct evidence for a gamma-glutamyl acyl ester on the enzyme. The absence of the acyl ester in a mutant carbamyl phosphate synthetase with a Cys269-->Ser substitution in the glutaminase subunit further suggests that the covalent intermediate is a thioester of Cys269. Under equilibrium conditions, the Cys269Ser mutant enzyme binds glutamine with a Kd of 7 +/- 1 microM, indicating that Cys269 is essential for acyl ester formation but not for binding of glutamine.


Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.


Annexin V forms calcium-dependent trimeric units on phospholipid vesicles.

The quaternary structure of annexin V, a calcium-dependent phospholipid binding protein, was investigated by chemical cross-linking. Calcium was found to induce the formation of trimers, hexamers, and higher aggregates only when anionic phospholipids were present. Oligomerization occurred under the same conditions annexin-vesicle binding. A model is proposed in which cell stimulation leads to calcium-induced organization of arrays of annexin V lining the inner membrane surface, thus altering properties such as permeability and fluidity.


Retinoic acid enhances the secretion of plasminogen from cultured rat microglia.

To determine the amount of plasminogen in microglial conditioned medium, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for rat plasminogen was established. Weak cross-reactivity with human serum plasminogen was observed, while no reactivity was detected with frog and carp plasminogen. The specificity of the immunosorbent assay was confirmed by Western blotting. The secretion of plasminogen into the microglial culture medium was quantified by using the established ELISA and was found to be increased depending on the culture time and number of microglia. The secretion was increased about 5-fold by stimulation with retinoic acid, while interleukin-1, and basic fibroblast growth factor showed no significant effect.


Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.


Identification of four FGF receptor genes in Medaka fish (Oryzias latipes).

Four types of cDNA clones encoding tyrosine kinases highly homologous to mammalian fibroblast growth factor receptors (FGF-R) were isolated from Medaka fish (Oryzias latipes) by the reverse transcription-polymerase chain reaction. Comparison of the four deduced amino acid sequences with four known mammalian FGF-Rs indicated that four FGF-R species corresponding to mammalian FGF-Rs exist universally in vertebrates including fishes, although FGF-R4 might have diverged sequences between fishes and mammals. Each of four FGF-R genes is transcribed to various extents as multiple mRNAs possibly by alternative splicing in adult fishes.


A mechanism for NADPH inhibition of catalase compound II formation.

Catalase-bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by 'endogenous' donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2-electron donors of the ethanol type, and from 1-electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one-electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2-electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.


Beta-glucosidase activity towards a bile acid glucoside in human liver.

Human liver contains hydrolytic activity toward 3 beta-glucosido-chenodeoxycholic acid. This beta-glucosidase activity, localized predominantly in the microsomal fraction, was optimally active in the presence of divalent metal ions close to pH 5.0 and was inhibited by EDTA. Kinetic parameters and other catalytic properties of hydrolytic activity towards 3 beta-glucosido-chenodeoxycholic acid from human liver microsomes are described.


cDNA sequence analysis of an antibiotic dodecapeptide from neutrophils.

The full-length cDNA of a neutrophil antibiotic dodecapeptide has been cloned by reverse transcription/PCR from bovine bone marrow RNA. This peptide was originally isolated from bovine neutrophils, and shown to exert a potent antimicrobial activity in vitro on both Escherichia coli and Staphylococcus aureus. The cDNA codes for a polypeptide of 155 amino acid residues with a predicted mass of 17,629 Da and a pI of 8.03. The deduced sequence comprises a putative signal peptide of 29 amino acids, a 114 residue pro-region, and a carboxy-terminal dodecapeptide corresponding to the mature antibiotic. The pro-sequence displays extensive identity to corresponding regions of other structurally unrelated antibiotic peptides of bovine neutrophils recently cloned.


Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.


Cytokines--involvement in reproduction.

To relay the current knowledge on the interaction between the immune and reproductive systems that results from sharing certain lymphohematopoietic cytokines and their receptors. Major studies related to this topic have been identified through MEDLINE searches and through the published literature. Those that have reported on the role of cytokines in the neuroendocrine events of reproduction, ovarian function, placenta, and the developing embryo. The field of growth factor and cytokines and their effects on reproduction is a rapidly growing new area of investigation. Immune cells and related cytokines have been shown to affect the neuroendocrine events of reproduction, ovarian function, placenta, and the developing embryo. Furthermore, it is now becoming apparent that these relationships are reciprocal in that the different cellular components of the neuroendocrine and reproductive systems and the developing embryo can modulate the production of cytokine by the immune system and can also produce certain cytokines. The presence of lymphocytes and macrophages in the female reproductive system, together with the fact that these cells may secrete soluble factors influencing embryo development and trophoblast growth, might suggest that cytokines may play a fundamental role in the mechanisms of immunological reproductive failure. In addition, different mixtures of these mediators, generated by immune cells, the developing embryo, or other maternal cells, may modulate the fine tuning of these activities. Current knowledge indicates a close interaction between the immune and reproductive functions. Further understanding of these interactions may lead to new concepts in fertility regulation.


Pituitary function before, during, and after chronic gonadotropin-releasing hormone agonist therapy.

To examine possible adverse effects on pituitary function of long-term administration of gonadotropin-releasing hormone agonist (GnRH-a). Prospective analysis of blood sampling before, during, and after GnRH-a therapy. Tertiary institutional outpatient care. Twelve normally ovulatory women with a diagnosis of endometriosis. Six-month suppression with GnRH-a. Serum levels of follicle-stimulating hormone, luteinizing hormone, free thyroxin index, cortisol (F), growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH). Basal and stimulated values of gonadotropins, PRL, F, TSH, and GH were normal and unchanged by 6 months of GnRH-a after resumption of menses. Utilizing dynamic pituitary function tests, we were unable to demonstrate an adverse effect of long-term GnRH-a therapy on pituitary function.


Effect of human corticotropin-releasing hormone on gonadotropin secretion in cycling and postmenopausal women.

To test the hypothesis that corticotropin-releasing hormone (CRH) is linked to stress-associated reproductive dysfunction in the human by determining if the administration of human corticotropin-releasing hormone (hCRH) results in an inhibition of gonadotropin secretion. Twenty-four-hour prospective study with frequent (every 10 minutes) blood sampling. University Clinical Research Center. Sequential 8-hour infusions of normal saline, hCRH (1 to 5 micrograms/kg per hour), and hCRH plus naloxone (2 mg/h). Four normal cycling women and four postmenopausal women. Plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and adrenal and ovarian steroids. In response to hCRH, a prompt and sustained rise in cortisol (F) was noted in both normal cycling women and postmenopausal women. No inhibition of LH or FSH was noted during either the hCRH or hCRH plus naloxone infusion in either group of women. Unexpectedly, elevations in the mean LH peak amplitude and the transverse mean LH concentration were noted in the postmenopausal women during the infusion of hCRH as compared with saline. The infusion of hCRH had no apparent effect on concentrations of PRL, FSH, and gonadal and adrenal steroids (except for F). Under these conditions, intravenously administered hCRH has no inhibitory effect on gonadotropin secretion in either premenopausal or postmenopausal women. The mechanism by which stress exerts its deleterious effect on reproductive function in the human remains unknown.


Influence of basal androgen levels in euandrogenic women on glucose homeostasis.

To evaluate possible relationships between insulin action and the normal variations of serum androgens in euandrogenic women. Prospective evaluation of insulin action in normal nonobese women using hyperglycemic and euglycemic hyperinsulinemic clamp techniques, correlating insulin action to serum testosterone (T), free T, androstenedione (A), and dehydroepiandrosterone sulfate (DHEAS). Statistical analysis used Spearman's rank correlation. Yale University Clinical Research Center. Nonobese females with normal oral glucose tolerance tests, on no medications known to affect glucose metabolism, having the following range of serum androgen levels: T, 0.69 to 3.12 nmol/L; free T, 0.17 to 1.25 nmol/L; A, 2.48 to 11.31 nmol/L; DHEAS, 0.68 to 10.61 mumol/L. Total number of patients studied: hyperglycemic clamps, n = 58; euglycemic hyperinsulinemic clamps, n = 43. None. Pancreatic insulin secretion in response to hyperglycemia and insulin action as assessed by insulin-mediated glucose utilization using the euglycemic, hyperinsulinemic clamp technique. We identified no significant correlation between serum androgens and either glucose uptake or insulin-mediated glucose utilization. Glucose-stimulated insulin release was negatively correlated with serum T and free T throughout the normal range of these hormones. We conclude that, within the normal range, variations of serum androgens are not correlated with changes in the response to insulin. It seems unlikely, therefore, that modest increases of serum androgens within the normal range are responsible for inducing insulin resistance.


Effect of hyperprolactinemia on the androgen response to an oral glucose load.

To investigate the effect of hyperprolactinemia on the androgen response to an oral glucose load in patients with normal and abnormal carbohydrate metabolism. Sixty-three women underwent a 75-g oral glucose tolerance test (OGTT) at approximately 6 weeks' postpartum. University hospital setting. Patients with a previous history of gestational diabetes. Adrenal and ovarian androgen response to oral glucose load. Results were stratified by the result of the OGTT (patients with impaired glucose tolerance or diabetes as a single group and patients with a normal OGTT as a separate group) and prolactin (PRL) level. All androgens decreased significantly over the 2-hour test in all groups except dehydroepiandrosterone sulfate (DHEAS) in the patients with both elevated PRL and abnormal glucose tolerance. Hyperprolactinemia and insulin resistance may act synergistically in the adrenal to alter DHEAS suppression and may result in chronically elevated circulating DHEAS levels.


Interinstitutional variability of follicle-stimulating hormone and estradiol levels.

To evaluate the variability of follicle stimulating hormone (FSH) and estradiol (E2) results from different laboratories. Prospective analyses of sera separated and frozen, obtained from 15 female patients in the follicular phase. All kits used for radioimmunoassay (RIA) for FSH were from the Second International Reference Preparation (IRP). Four different kits were used for FSH and three for E2. Sera obtained from each patient were separated into five test tubes and frozen. Analysis of all samples was done on the same day in each one of five participating hospital-based RIA laboratories (North Shore, Cornell, Yale, Mount Sinai, and Norfolk). Fifteen consecutive patients from the assisted reproductive technology program at North Shore University Hospital participated in the study. After FSH and E2 levels were tabulated for each laboratory, mean levels were calculated. Using the Bonferroni adjusted pairwise multiple comparisons analysis, significant differences were found between three groups of laboratories within the FSH results and three different groups within the E2 results. [1] Different results may be obtained on the same sera for FSH and E2 at different laboratories; [2] for FSH, even though the same IRP is used, different results are reached; [3] for E2, even when the same kits are used, results may be significantly different. These results suggest that specific FSH and E2 levels used to predict chances for achieving a viable pregnancy through in vitro fertilization should be interpreted with caution across institutions.


Endometrial dating in the conception cycle.

To investigate endometrial dating during the conception cycle. Endometrial biopsies of the last half of the luteal phase of conception cycles were dated based on urinary luteinizing hormone (LH) surges. Endometrial samples were obtained from women attending the fertility clinic of the Department of Obstetrics and Gynecology at Gunma University Hospital. One hundred eighty-five women, of whom 15 (8.1%) conceived during this study. Urinary LH surge, serum progesterone (P) levels, and endometrial dating. All 10 women who conceived showed 2 or within 2 days in-phase endometrial biopsies on days 7 to 11 after the LH surge. In 4 of 5 women biopsied on day 12, an unequivocally delay in the stroma was found, i.e., a persistence of edema and poor development of predecidual reaction. Since serum P levels in conception cycles were significantly higher than in nonconception cycles on days 10, 11, and 12, we interpreted this delay in the stroma as a consequence of conception. Endometrial specimens during the last half of the luteal phase of conception cycles are in-phase until day 12. On day 12, gestational hyperplasia causes apparent out-of-phase.


Transcervical cannulation of the fallopian tube for the management of ectopic pregnancy: prospective multicenter study.

To determine the efficacy of transcervical tubal cannulation and intraluminal methotrexate injection for the management of tubal ectopic pregnancy (EP). Prospective multicenter study of 33 patients with tubal pregnancies. Four university-based gynecology and radiology departments in three different countries: France, England, and Germany. Thirty-three patients who presented with a clinical diagnosis of EP. Patients underwent transcervical tubal cannulation under fluoroscopic or ultrasound control and local injection of methotrexate (up to 50 mg). We evaluate the feasibility of transcervical tubal cannulation for the management of tubal pregnancy. Two patients elected to withdraw from the protocol. In the remaining 31 patients there was complete resolution of the EP in 27 (87%). Surgery was performed in 4 patients. Seventeen patients, 14 of whom desired pregnancies, were available for follow-up to assess the return of reproductive potential. Seven of 7 patients who subsequently underwent hysterosalpingography had patency of the affected tube. Five patients later had an intrauterine pregnancy. One patient had an early miscarriage, two have given birth, and two singleton pregnancies are still ongoing. The remaining patients are symptom free. This study demonstrates that transcervical tubal catheterization in patients with tubal pregnancies is feasible and can be performed without anesthesia or analgesia in most cases. Intraluminal methotrexate per se is capable of causing regression of the EP. This approach offers a new alternative for the treatment of selected patients with tubal EP.


Standardization of hysterosalpingography and selective salpingography: a valuable adjunct to simple opacification studies.

To investigate the correlation between opacification and perfusion pressures during hysterosalpingography (HSG) and selective salpingography under the assumption that the latter may add to the diagnostic capabilities of the procedures. Perfusion pressures were uniformly evaluated by standardizing injection volume per time interval of contrast medium and the delivery system. Pressures were measured in a closed system through a digital manometer and recorded on tracing paper. Fully ambulatory gynecoradiology suite at academically affiliated infertility center. Thirty infertility patients. Hysterosalpingography and selective salpingography for diagnostic purposes. Correlation between opacification patterns and perfusion pressures. The evaluation of perfusion pressures during HSG is unreliable because they may be affected by uterine factors and will only reflect the oviduct of least resistance. In contrast, perfusion pressures during selective salpingography are reflective of only the investigated tube. They appear to lie within a functionally normal range of up to 350 mm Hg. Tubes by opacification judged as normal exhibited a pressure range of 429 +/- 376 mm Hg, which was significantly lower than that of abnormally appearing oviducts (957 +/- 445 mm Hg; P = 0.001). The concomitant performance of perfusion pressure studies during selective salpingography further enhances the diagnostic capability of selective salpingography over HSG in the diagnostic evaluation of fallopian tubes.


A contraceptive subdermal implant releasing the progestin ST-1435: ovarian function, bleeding patterns, and side effects.

To study ovarian function, bleeding patterns, and side effects during the 1-year use of a new modified contraceptive subdermal implant releasing the progestin ST-1435 with a lifetime of 2 years. The effect on ovarian function and bleeding patterns of one contraceptive implant releasing the progestin ST-1435 was studied in 26 healthy women who volunteered. Side effects were recorded. The outpatient clinic of the City Maternity Hospital, Helsinki, Finland. One ST-1435 contraceptive implant was inserted subcutaneously into the ventral aspect of left upper arm. The women attended the clinic at half-year intervals. Records of bleeding were kept. Blood samples were collected from 5 women before insertion of an implant, from 12 women during the first 5 to 6 weeks of use, and from 10 women during the 6th and 12th month of use. Serum concentrations of ST-1435, progesterone, and estradiol were determined. Side effects were reported. The study covered 302 woman-months. The implant gave serum concentrations of ST-1435 high enough to inhibit ovulation in all of the 37 analyzed cycles. No pregnancies occurred. Irregular bleeding or spotting was the main event observed, especially during the 1st year of use. One half of the users had irregular cycles. None of the women's implants was removed during 1 year of use because of irregular bleeding. The implant was well accepted and tolerated by the women; no hormonal side effects were reported. One single 4-cm subdermal ST-1435 implant with a lifetime of 2 years showed good contraceptive efficacy and led to suppression of ovulation. No hormonal side effects were reported. Irregular bleeding patterns were common but well-tolerated, and the implant had a high continuation rate.


In vitro lymphocyte activity in women with endometriosis--an altered immune response?

To determine the possible role of the immune system in the pathogenesis of endometriosis. The lymphocyte proliferative response in the presence of autologous endometrial cells was assayed by tritiated thymidine incorporation. Patients were recruited from a university outpatient clinic. To determine the lymphocyte proliferative response to endometrium in controls and patients with endometriosis. Twenty patients with endometriosis and 26 control women were studied. The lymphocyte proliferative response in the presence of autologous endometrium was significantly lower in women with endometriosis when compared with controls. This study indicates that an altered lymphocyte/endometrial cell relationship is operational in women with endometriosis and may contribute to the pathogenesis of the disease.


Steroidogenesis of cultured granulosa cells in women at risk for ovarian hyperstimulation syndrome.

To determine if cultured human granulosa cells (GCs) obtained from women at risk for ovarian hyperstimulation syndrome (OHSS) possess altered steroidogenic capacity. Prospective analysis of 28 consecutive in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) cycles. In Vitro Fertilization Program at Rush Presbyterian St. Luke's Medical Center in Chicago, Illinois. Eighteen patients (group I) with serum estradiol (E2) levels > 7,342 pmol/L on the day of exogenous human chorionic gonadotropin (hCG) administration (day 0) with > 10 ovarian follicles present (high risk for OHSS); 10 patients (group II) with E2 < or = 7,342 pmol/L on day 0 and < or = 10 follicles. Human GCs obtained during gonadotropin-releasing hormone agonist-pretreated IVF-GIFT cycles were cultured in the absence (control) or presence (hCG) of hCG, 1 IU/mL, and/or androstenedione (A) 10(-7) M. Granulosa cells obtained from follicles < or = 15 mm diameter were cultured separately from those obtained from follicles > 15 mm diameter. Estradiol (E2) and progesterone were measured in tissue-culture medium by a solid-phase direct radioimmunoassay. In vitro E2 production by cultured GCs was significantly increased in follicles < or = 15 mm diameter from women considered at risk of developing OHSS (group I). Estradiol response to hCG and/or A appeared enhanced in all follicles in group I. Progesterone production in the basal and hCG challenged state was greater in cells obtained from large follicles in group I than in group II. Ovarian hyperstimulation syndrome appears to be a function of an increased number of follicles that express an enhanced steroidogenic capacity.


The prevalence and predictability of depression in infertile women.

To determine the prevalence, severity, and predictability of depression in infertile women compared with a control sample of healthy women. Subjects were assessed while waiting to see their physician: infertility patients before a visit with an infertility specialist and control subjects before seeing either a gynecologist or internist for a routine gynecological examination. Subjects completed a demographic form and two depression scales. A group infertility practice affiliated with an academic medical center, a hospital-based gynecology practice, and a health maintenance organization internal medicine clinic. 338 infertile women and 39 healthy women. None. The Beck Depression Inventory and the Center for Epidemiological Studies Depression Scale. The infertile women had significantly higher depression scores and twice the prevalence of depression than the controls; women with a 2- to 3-year history of infertility had significantly higher depression scores compared with women with infertility durations of < 1 year or > 6 years; women with an identified causative factor for their infertility had significantly higher depression scores than women with unexplained or undiagnosed infertility. Depressive symptoms are common in infertile women. Psychological interventions aimed at reducing depressive symptoms need to be implemented, especially for women with a definitive diagnosis and for those with durations of 2 to 3 years of infertility.


Ultrashort gonadotropin-releasing hormone agonist (GnRH-a) protocol in comparison with the long-acting GnRH-a protocol and menotropin alone.

To compare the in vitro fertilization and embryo transfer (IVF-ET) outcome of a 3-day gonadotropin-releasing hormone agonist (GnRH-a) administration: ultrashort protocol with the outcome of long-acting GnRH-a cycles or human menopausal gonadotropin (hMG) alone. Ninety-two cycles of the ultrashort protocol were matched with 92 cycles with long GnRH-a and with 92 hMG cycles. The IVF-ET program. Amount and duration of hMG treatment, hormonal profile on the day of human chorionic gonadotropin administration, cancellation rate, number of oocytes retrieved, and fertilization and pregnancy rates (PRs) were examined and compared among the three groups. The ultrashort group needed a higher number of hMG ampules than the hMG group but significantly less than in the long GnRH-a regimen. The number of oocytes in the ultrashort protocol was the same as in the long GnRH-a, but the number of embryos per retrieval was significantly lower than with the long GnRH-a protocol and similar to that found in the hMG group. The ultrashort protocol yielded 10% PR per cycle and 17% per replacement, significantly lower than with the long GnRH-a protocol, 26% and 36%, respectively, but also lower than in the hMG one, namely 13% and 28%. The ultrashort protocol, although being convenient and having some advantages found in the long GnRH-a protocol, is inferior in its outcome compared with the two other protocols.


Prospective study of short and ultrashort regimens of gonadotropin-releasing hormone agonist in an in vitro fertilization program.

To assess the usefulness of the ultrashort regimen of gonadotropin-releasing hormone agonist (GnRH-a) in ovulation induction in an in vitro fertilization (IVF) program. A prospective randomized trial comparing short and ultrashort regimens of GnRH-a. Aberdeen Assisted Reproduction Unit. Forty-eight patients having IVF for the first time were randomized between the two protocols. Response to ovarian stimulation and occurrence of spontaneous luteinizing hormone (LH) surges. In ovulation induction, fertilization, and pregnancy rates the ultrashort regimen produces results that were no different to the short regimen but it did not always prevent an LH surge. The ultrashort regimen can be a useful alternative for ovarian stimulation of patients undergoing IVF.


Influence of ovarian cysts on the results of in vitro fertilization.

To determine if ovarian cysts are associated with a particular basal endocrine profile and impair follicular growth. Retrospective study. In Vitro Fertilization (IVF) Center. Nine hundred fourteen stimulation cycles stimulated with a combination of luteinizing hormone-releasing hormone analogues (LH-RH-a) and human menopausal gonadotropins (hMG) in a long protocol in an IVF program. After 15 days of LH-RH-a therapy, ovarian cysts (> or = 20 mm) were observed in 8% of cases. These cysts were not related to a particular basal endocrine profile and did not impair follicular growth and IVF results. However, puncturing these cysts enhanced the quality of subsequent follicular growth. On the contrary, cysts appearing during hMG treatment (> or = 25 mm) were related with a lower LH:follicle-stimulating hormone ratio (0.79 +/- 0.52 versus 0.92 +/- 0.74 in absence of cyst) and to a lower ovarian response as assessed by the maximal estradiol level to the total number of hMG ampules ratio (51.6 +/- 36.5 versus 65.9 +/- 47.9 in absence of cyst). However, this difference had no influence on the pregnancy per stimulation rate (18% versus 16% in absence of cyst; not significant). Results show that the pathogens of ovarian cysts appearing during the blockage phase and during the stimulation phase are different. However, they do not impair the results of IVF, and thus it is not necessary to cancel the attempt in case of ovarian cyst.


Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction.

To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. University-affiliated tertiary care center. Normal healthy volunteers. Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. HA and acrosome reaction. Sperm HA was significantly increased at 10 minutes by P both at 10(-7) M (9.27 +/- 1.59%; mean +/- SEM) and 10(-6) M (9.39 +/- 1.94%) when compared with untreated spermatozoa (5.62 +/- 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10(-6) M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10(-6) M to P at 10(-7) M (12.43 +/- 3.31%) or P at 10(-6) M (13.52 +/- 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.


Correlation between puberty and the development of autoimmunity to spermatozoa in men with cystic fibrosis.

To test the hypothesis that puberty is a necessary factor in the pathogenesis of autoimmunity to sperm in men with cystic fibrosis (CF), we studied prepubertal and postpubertal males with CF versus an age-matched group of males with type 1 diabetes as controls. Sera from CF and diabetic males treated at University Hospital, State University of New York, Stony Brook, were tested by indirect immunobead binding for antisperm antibodies and by radioimmunoassay for testosterone (T), luteinizing hormone, and follicle-stimulating hormone. The finding of autoantibodies to spermatozoa was correlated with chronological age, as well as with clinical and hormonal pubertal status. Autoimmunity to sperm, as detected by humoral antisperm antibodies, was documented solely in postpubertal males, as judged by hormonal and clinical criteria. Eighty-three percent of sexually mature CF males and 6.3% (1 of 16) diabetic males exhibited autoantibodies to sperm. These antibodies were only detected when serum T levels were > 8.7 nmol/L (250 ng/dL). These results suggest that puberty, and presumably, active spermatogenesis is a requirement for the development of autoimmunity to sperm in men with CF.


Enhanced gamete interaction in the sperm penetration assay after coincubation with pentoxifylline and human follicular fluid.

To evaluate the effect of pentoxifylline and heat-inactivated human follicular fluid (FF) on performance in the sperm penetration assay (SPA) as a paradigm for the effect of these agents on human sperm-egg interaction in vivo and in vitro fertilization. Semen specimens from men undergoing SPA testing for evaluation of suspected male factor infertility were coincubated with neat medium or media supplemented with pentoxifylline or human FF in a nonblinded manner. Twenty male factor infertility patients. Semen specimens were preincubated with: [1] pentoxifylline 0.25 mg/mL; [2] 10% human FF; [3] pentoxifylline+human FF; and [4] neat Biggers, Whitten, and Whittingham medium. Differences in the rate of penetration of zona-free hamster oocytes. Preincubation with either human FF or pentoxifylline produced a significant improvement in hamster egg penetration rates. Coincubation with a combination of human FF and pentoxifylline resulted in a significant enhancement of penetration as compared with single agent treatment. Coincubation of sperm with human FF and pentoxifylline may provide a means of enhancing sperm activity for insemination and assisted reproduction.


Interval required for gonadotropin-releasing hormone-agonist-induced down regulation of the pituitary in cynomolgus monkeys and duration of the refractory state.

To determine the minimal interval of gonadotropin-releasing hormone agonist (GnRH-a) administration required to induce pituitary refractoriness to the positive feedback effects of estradiol (E2) and to determine the duration of the induced refractory state in a nonhuman primate model. Research laboratories of The Jones Institute for Reproductive Medicine. Cynomolgus monkeys with documented regular menstrual cycles. Part 1. Groups of four monkeys treated with leuprolide acetate (LA) for increasing intervals (0, 2, 4, 6, and 8 days), then challenged with E2 benzoate to induce a gonadotropin surge. Part 2. Groups of four monkeys treated with LA for the minimal time required to induce pituitary refractoriness (6 days), then challenged with E2 benzoate 2, 4, or 7 days after cessation of LA treatment. Pituitary luteinizing hormone and follicle-stimulating hormone and E2 levels were monitored. A minimum of 5 days of treatment is required for LA to induce pituitary refractoriness to the positive feedback effects of E2 benzoate. Once the down regulated condition is achieved, the refractory state lasts for at least 4 but not more than 6 days. The minimal treatment interval required for a GnRH-a to induce pituitary refractoriness to the positive feedback effects of estrogen and the duration of the induced refractory state are determined to be 5 days for these primates.


Experimentally induced endometriosis in rats: effect on fertility and the effects of pregnancy and lactation on the ectopic endometrial tissue.

To investigate the repercussion of experimentally induced endometriosis on the fertility of Wistar rats and the effects of pregnancy and lactation on endometrial ectopic implants. Eight-five animals were used for the experiment. Experimental endometriosis was induced in 44 and the remaining 41 were sham-operated (control group). After evaluating the growth of the implants in a second laparotomy, all the animals were mated until finding sperm in vaginal smears. In third and fourth laparotomies during pregnancy and lactation, pregnancy rate was determined, and the implants were re-evaluated, being resected for histological study. Serum estradiol (E2) and progesterone (P) levels were also measured. The pregnancy rate in animals with endometriosis was 65.7% versus 100% in control group. No statistical differences in days of gestation and number of pups were found between the two groups. Ectopic implants underwent a significant regression during lactation period. Serum E2 and P levels and histological features sustained these findings. Experimentally induced endometriosis diminishes pregnancy rate in rats. It is not pregnancy, but the anestrus condition created by lactation that exerts a beneficial effect on experimentally induced endometriosis in rats.


Proliferative and morphogenic changes induced by the coculture of rat uterine and peritoneal cells: a cell culture model for endometriosis.

To evaluate the proliferative and morphogenic effects induced by the coculture of uterine and peritoneal cells to establish a cell culture model for endometriosis. Uterine epithelial and stromal cells and peritoneal mesothelial and subserosal cells were cocultured with homologous cell types, heterologous cell types, or as isolated populations using a bicameral chamber design. Department of Obstetrics and Gynecology at the University of Missouri, Columbia, Missouri. Cells isolated and purified from five mature female Sprague Dawley rats of normal reproductive status were used to establish cell cultures. Cell proliferation (deoxyribonucleic acid synthesis) was measured by the incorporation of 3H-thymidine, and cell morphology was assessed using inverted phase-contrast microscopy. Peritoneal mesothelial cells augmented proliferation and induced cellular aggregation of uterine stromal cell monolayers. Peritoneal subserosal cells amplified proliferation and induced an irregular, compacted morphology in uterine epithelial cells. The proliferation and morphology of the two peritoneal cell types was not altered by uterine cell coculture. The coculture of uterine and peritoneal cells in bicameral chambers provides a tool to study the paracrine interactions of cells that comprise the endometriotic lesion. The altered proliferation and morphology of the uterine cells may be related to the histologic and biochemical asynchrony observed between uterine endometrium and ectopic endometriotic tissue in vivo and offers insight into possible mechanisms of the histogenesis of endometriosis.


The menstrual cycle: relations of biophysical and hormonal determinations in normal women of reproductive age.

Appropriate relationships between circulating levels of 17 beta-E2 and P are necessary for normal endometrial development and blastocyst implantation. The aim of this study was to relate biophysical and biochemical measurements obtained during the menstrual cycle of six healthy women in 25 menstrual cycles. One hundred eighteen vaginosonographic determinations of the endometrial thickness together with serum E2 and P assays were performed at 5-day intervals. The three parameters studied were standardized by the formula: observation minus mean determination for that parameter divided by the standard deviation of that parameter. Using this common unit, the physiological relations between ultrasonographically determined endometrial thickness, E2, and P were readily seen.


The role of office hysteroscopy in in vitro fertilization.

Twenty-eight patients participated prospectively in a study to evaluate the impact of hysteroscopically detected uterine and cervical anomalies on the success rate of ET in an IVF-ET program. All participants had a normal intrauterine cavity by standard HSG. All the patients had a diagnostic office hysteroscopy under paracervical block before commencing COH. Because our IVF program does not include hysteroscopy as a requirement before undergoing IVF and because the significance of mild intrauterine abnormalities is not yet known, the hysteroscopic findings were not relayed to the personnel involved in the IVF-ET procedure. Sixteen patients (group I) had a normal hysteroscopic evaluation. Twelve patients (group II) had abnormal hysteroscopic findings including small uterine septa, small submucous fibroids, uterine hypoplasia and cervical ridges. Although no difference in patients or cycle characteristics was present, there was a significant difference in the clinical PR between patients in groups I and II. In conclusion, in an IVF-ET program patients with normal hysterography but abnormal hysteroscopic findings had a significantly lower clinical PR, demonstrating the importance of performing hysteroscopy before IVF-ET.


Local anesthesia with conscious sedation for laparoscopic intrafallopian transfer.

Local anesthesia with conscious sedation is well accepted by patients and provides scheduling flexibility, cost containment, patient safety, and ease of recovery. We believe the technique should be offered to selected patients undergoing intrafallopian transfer. By adhering to specific guidelines for surgical technique and monitoring, the procedure is a safe and acceptable alternative to general anesthesia for laparoscopic intrafallopian transfers.


Method for the transportation of peritoneal macrophages.

This report outlines a method of PF transport that preserves the viability and activation level of peritoneal macrophages. Collection of macrophages in this manner will allow transport of PF from multiple centers for evaluation of macrophage function in a single laboratory. Such technology will make it possible to intensively study the relationship between peritoneal macrophages and infertility.


Chlamydia antibody by enzyme-linked immunosorbent assay and associated severity of tubal factor infertility.

We have evaluated the clinical utility of the serum chlamydial antibody value by ELISA in predicting the severity of tubal factor infertility quantified by the use of a previously reported pelvic scoring system. Significant differences in salpingitis scores, tubal occlusion scores, adhesion scores, and pelvic stages were found with increasing chlamydia antibody grades. The level of ELISA value strongly correlated with salpingitis, tubal occlusion, and total pelvic scores and pelvic stages. The serum chlamydia antibody level by ELISA now appears to be a useful prognostic tool for predicting the severity of tubal factor infertility in addition to its value in screening for its presence.


Umbilical metastases from a stage IB cervical cancer after laparoscopy: a case report.

The first reported case of laparoscopy site metastases from an unsuspected stage IB cervical cancer diagnosed during laparoscopy for endometriosis is presented. Implications of this clinical situation are discussed.


Fresh and frozen-thawed human sperm bind in a similar pattern to the zona pellucida in the hemizona assay.

This study evaluated the impact of sperm cryopreservation on sperm quality. The HZA was used to test the binding capacity of fresh as opposed to frozen-thawed sperm from 12 donors. Fresh and frozen-thawed sperm motility was 47% +/- 1.5% and 24% +/- 3.8% (mean +/- SE), respectively. However, the number of sperm cells attached to the hemizonae was 75 +/- 12.0 and 74 +/- 11.9, respectively. We conclude that cryopreservation results in a reduced number of motile sperm cells but does not adversely affect the ability of rescued sperm cells to bind to the ZP. The study also supports the use of frozen-thawed rather than fresh donor sperm for control in the HZA procedure.


Round cells and sperm fertilizing capacity: the presence of immature germ cells but not seminal leukocytes are associated with reduced success of in vitro fertilization.

Fifty semen samples produced for IVF by patients diagnosed as having unexplained infertility were screened for leukocytes, leukocyte subsets, and immature germ cells using a mAB-based staining procedure. Nonfertilizing ejaculates were found to contain significantly larger numbers of immature germ cells, although no significant differences in leukocyte content were observed between groups. Neither sperm density or progressive motility were significantly different between fertilizing and nonfertilizing groups. We conclude that seminal leukocytes have little if any influence on the fertilizing capacity of the spermatozoa in patients undergoing IVF for unexplained infertility, but the presence of large numbers of germinal elements is associated with reduced fertilizing capacity and may be indicative of an immature sperm population.


Laparoscopic internal spermatic vein ligation: report of a new technique.

Over the past 22 months, 51 laparoscopic internal vein ligations have been performed to determine its application and practicality in treating the infertile male with varicoceles. Of the 33 cases available for a follow-up of greater than 6 months, 16 pregnancies are reported. Five patients reporting pregnancies refused to submit a postoperative semenogram. Of the reportable series, 17 of 33 had significant improvement in sperm density (51%), 15 of 33 (45%) in sperm viability, and 15 of 33 (45%) in sperm motility. No major complications were seen and minor complications were few and transient. Morbidity was extremely low. Laparoscopic internal spermatic vein ligations would appear to be a reasonable, practical, and effective method to correct varicoceles.


[Did growth hormone-deficient children grow up to normal adult height by growth hormone treatment?].

One of the important aims of growth hormone (GH) treatment in GH deficient children is to allow them to grow to their full genetic potential. Usually, however, the final height of GH deficient children does not reach normal adult height and is below their target height. Furthermore, isolated GHD with spontaneous puberty is known to lead to a shorter adult height than that obtained in GHD associated with gonadotropin deficiency. The height at the start of puberty is reported to be well correlated with final height in GHD. Therefore, when isolated GHD subjects treated with GH reached puberty while they were still shorter than normal, they ended up by being shorter than normal as adults. The trial to increase the GH dose during puberty did not seem to increase the final height. The gonadal suppression therapy combined with GH treatment significantly increased the final height in isolated GHD. It is now the consensus that insufficient height at the onset of puberty leads to short final height and that early diagnosis of GHD is thus important to allow catch-up growth to optimal height before puberty. It may also be beneficial to treat GHD with higher doses to overcome the waning phenomenon with GH treatment.


[Difference of acute ketone body metabolism between insulin-dependent diabetic and non-insulin-dependent diabetic individuals].

The difference in the acute metabolic change in ketone bodies between patients with insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) was investigated in this study. The subjects employed were 7 patients with IDDM losing residual insulin secretion and 7 patients with NIDDM matched to the former patients for age, body mass index, duration of diabetes, daily insulin dosage, fasting plasma glucose and HbA1c. Blood samples were drawn at 3A.M. and 7A.M. on the same day, and plasma glucose, acetoacetic acid (AcAc), 3-beta-hydroxybutylic acid (3-OHBA), free fatty acid (FFA), glycerol, cortisol and growth hormone (GH) concentrations were determined. Plasma total ketone bodies (AcAc and 3-OHBA), 3-OHBA and FFA concentrations at 7A.M. were significantly higher in the patients with IDDM than in those with NIDDM (p < 0.05), while there were no significant differences in any other parameters at 3A.M. between the patients with IDDM and those with NIDDM. The ratios of 7A.M. value/3A.M. value of total ketone bodies, AcAc and 3-OHBA concentrations were also more significantly elevated in the patients with IDDM than in those with NIDDM. It was observed that the ratio of 3-OHBA was more than 2.0 in all of the patients with IDDM and less than 2.0 in all of the patients with NIDDM, the difference being significant with p < 0.001.(ABSTRACT TRUNCATED AT 250 WORDS)


[Pregnancy in Cushing's syndrome].

Pregnancy in cases of Cushing's syndrome is rare. A pregnant patient with Cushing's syndrome due to an adrenal adenoma who was diagnosed in the third trimester is described. She underwent conservative treatment for Cushing's syndrome and delivered a normal infant by Caesarean section. Currently, 121 pregnancies in 97 patients have been reported, but a principle for the treatment of the mother and fetus has not yet been established. We reviewed pregnancy in Cushing's syndrome based on the world literature and evaluated the choice of treatment to take. In the first trimester of pregnancy, therapeutic abortion or surgical treatment, such as adrenalectomy or resection of the pituitary tumor in Cushing's syndrome, is recommended for patients with severe hypercorticism (plasma cortisol > or = 30 micrograms/dl, urinary 17-OHCS > or = 15 mg/day, urinary free cortisol > or = 1000 micrograms/day), while conservative treatment is recommended for patients with mild hypercorticism (plasma cortisol < 30 micrograms/dl, urinary 17-OHCS < 15 mg/day, urinary free cortisol < 1000 micrograms/day). In the second trimester of pregnancy, surgical treatment is recommended for patients with severe hypercorticism, while conservative treatment is recommended for patients with mild hypercorticism. In the third trimester of pregnancy, Caesarean section is recommended for most cases. Drug treatments such as with metyrapone should be limited to patients showing severe hypercorticism or a maternal high risk who have contraindications to surgical treatment.


Epidemic of malaria in Barmer district (Thar desert) of Rajasthan during 1990.

Barmer district of Rajasthan in Thar desert and hitherto a hypoendemic area for malaria came in the grip of a severe malaria epidemic during 1990. The epidemic occurred as an aftermath of floods, preceded by normal rains during 1988 and 1989 after a prolonged drought phase. The epidemic was spread over the whole district including Barmer town. Annual Parasite Incidence (API) and Annual falciparum Incidence (AFI) for the district touched record figures of 17.20 and 5.83 respectively while for the Barmer town they were 36.5 and 14.0 respectively. Out of the eight PHCs, Baitu PHC was the worst affected where the two indices touched all-time high figures of 55.3 and 19.6 per cent respectively. A total of 122 infants were reported positive for malaria, of which 103 were contributed by Baitu PHC alone. Eighty per cent infant positivity was spread over September and November, indicating a high rate of transmission. A total of 47 deaths due to cerebral malaria were reported. However, in view of the high infectivity among infants and paediatric groups there was a strong possibility of deaths among these groups which could not be verified. Entomological findings revealed that a sudden increase in An. culicifacies densities due to extensive breeding potential, as a sequel to floods, activated the transmission, which was maintained at a low level by An. stephensi, predominant in this desert region. Other factors which contributed to the intensity and extent of epidemic were the return of drought-migrated population from malarious areas, low cattle density, malaria non-immune population, inadequate and poor spray coverage and delayed radical treatment. Insecticide adult susceptibility tests revealed a high degree of resistance in An. stephensi against DDT and dieldrin.


Gujarat model of health management information system with reference to malaria.

The Gujarat model of health management and information system has been developed with the objective of reducing the work load of health workers at all levels pertaining to record keeping and report preparation for enhancing the quality of health services, as well as for better implementation and timely monitoring of health programmes. Pro formas currently in use under various health programmes have been reorganized and updated. For monitoring malaria, the total number of registers has been reduced from seven to one, reporting forms from 23 to six and the number of columns in reporting forms from 493 to 59. The model is expected to save the working time of laboratory technicians (15%) and multi-purpose health supervisors (5%) at PHC level and of all workers (100%) engaged in report preparation at district, state and national levels. The data generated by using this model are expected to be of high quality and accuracy and should lead to more rational planning.


Antigenic diversity amongst ten geographic isolates of Plasmodium falciparum defined by merozoite invasion inhibition assay.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of human malaria parasite Plasmodium falciparum will influence the design of vaccine against malaria. In this study, in vitro inhibition of merozoite invasion in erythrocytes by an immune human serum was used to define the antigenic differences in 10 isolates of P. falciparum from three endemic areas, i.e. Africa, South America and Southeast Asia. The serum inhibited the invasion of merozoites of all the strains but the extent of inhibition varied from low to moderate to high degree indicating antigenic differences amongst isolates of P. falciparum. The antigenic differences could not be correlated to the geographic origin of the parasite isolate.


Observations on malaria patients seeking treatment in hospitals in a rural and an urban area of Sri Lanka.

Malaria in Sri Lanka is endemic in the dry zone and occurs during epidemics in the wet zone. A survey was carried out on malaria patients presenting at a hospital located in a predominantly rural area in the dry zone (Polonnaruwa) and an urban area in the wet zone (Ragama). Higher incidence of Plasmodium falciparum infections than reported nationally were observed in both locations. Of particular interest is the rapidity with which patients gained access to hospital treatment after the onset of malaria symptoms. The observed mode is 3-6 days. It is postulated that early treatment may impair the development of clinical immunity to malaria in the Sri Lankan population.


Breeding habitats and their contribution to Anopheles stephensi in Panaji.

A one-year longitudinal study conducted in 9 categories of breeding habitats in Panaji, Goa, showed that 1.1% of the 67,360 breeding sites contained Anopheles stephensi immatures and the overall positivity varied from 0.4 to 3.5% with a peak in June. The habitat-wise proportion of An. stephensi was: wells, 0-1.3%; fountains, 1.4-11.4%; masonry tanks, 0.8-6.1%; overhead tanks, 0.1-4.0%; curing water in construction sites, 0.6-9.0%; groundwater tanks, 0-1.4%; tyres, 0-8.9%; barrels and tins, 0-5.4%; and intradomestic containers, 0-1.9%. An. stephensi was breeding along with An. subpictus, An. vagus, An. barbirostris, Culex quinquefasciatus, Cx. vishnui, Ae. aegypti, Ae. albopictus and Ae. vittatus.


Reversal of chloroquine resistance with verapamil in P. berghei in vivo.

The effects of verapamil on the parasite susceptibility to chloroquine were examined in mice infected with chloroquine-sensitive and chloroquine-resistant lines of Plasmodium berghei. Verapamil in a dose of 10-50 mg/kg daily s.c. for 4 days did not affect the growth of both sensitive and resistant parasites. When verapamil in the same dose range was combined with 1.5 mg/kg chloroquine diphosphate, the chloroquine-sensitive parasites became more susceptible to chloroquine. Similarly, verapamil severely suppressed the growth of chloroquine-resistant parasites in combination with 3 mg/kg (base) of chloroquine, but the reversal of resistance was not complete. Thus, still higher doses of verapamil, which are not tolerated by the host, are required for the complete reversal of resistance.


Biochemical alterations in Plasmodium vivax-infected malarial patients before and after radical treatment.

Biochemical alterations in 152 malaria patients infected with Plasmodium vivax were studied and the effect of parasitaemia on these changes was assessed. The degree of parasitaemia correlated positively with plasma uric acid, total and unconjugated bilirubin. A decrease in the levels of serum total protein, albumin, serum total, free and ester cholesterol was observed in vivax malaria. A follow-up study done on a section of the above patients after administration of chloroquine and primaquine for radical treatment of malaria showed the most of the alterations observed were bought back to normal. However, blood haemoglobin level was not restored to normal even after ten days of commencement of treatment.


Gambusia affinis: dispersal due to floods and its failure to colonize new water bodies in Shahjahanpur District (U.P.).

In villages of District Shahjahanpur, 122 decentralized Gambusia multiplication ponds were established to cover the need of the entire district. Profuse breeding of Gambusia was observed in these ponds. The fishes are being successfully used in mosquito control all over the district. In July 1990 there was a widespread flood due to which 70 Gambusia multiplication ponds were affected and the fish was washed away in large numbers, leaving only a scanty population in the flood-affected ponds. We utilized this opportunity to study the natural dispersal and colonization of Gambusia in different aquatic habitats. The study revealed that Gambusia was either not found in most habitats or was present in very small numbers, and on its own Gambusia was unable to eliminate the local fauna to become a dominant species. Predatory fishes and birds played a major role in eliminating Gambusia. Gambusia is therefore unlikely to pose any ecological hazard in vector-control programmes.


Some observations on Plasmodium falciparum gametocytaemia in natural infections in an endemic area of Koraput district, Orissa.

Peripheral P. falciparum gametocytaemia with respect to its minimum average time taken for appearance in peripheral blood, its peak density, duration of persistence in peripheral circulation, conversion rate and sex ratio was studied in 22 persons with natural infections in a village of Koraput district, Orissa. The minimum average time taken for the appearance of gametocytes was 9.3 days in children (below 15 years) and 8.9 days in adults (15 years and above). The peak count was low, the maximum density being 31/microliters. The duration of gametocytaemia in peripheral circulation was also short, the longest being 32 days. The gametocyte conversion rate on an average was 0.74% in children and 2.04% in adults. The mean sex ratio of micro:macro gametocytes was 1:2.6 in children and 1:6.2 in adults.


Effect of tissue culture media on multiplication of Plasmodium falciparum in vitro.

To date RPMI-1640 has been the best medium for cultivation of Plasmodium falciparum in vitro. In addition to this medium, several alternative media, essentially the ones used in animal and plant tissue culture, were employed for the cultivation of P. falciparum. Only the media rich in glucose content, viz. Nitsch medium and White's medium S-3, supported the parasite multiplication.


Microscopic diagnosis of malaria in Kheda district of Gujarat.

A study conducted in 1990 revealed that 2% (range 0.6-4.8) of negative blood smears were mislabelled as positive, and 6.7% of positive blood smears were mislabelled as negative. A result of such mislabelling would be inadequate treatment of a large number of patients. Hence the need to look into the training aspect and system of supervision of laboratory technicians. The present system of cross-checking of blood smears at different levels also needs to be reviewed. A study which could address itself to these needs is indicated.


Problem of antimalarial drug resistance in Plasmodium falciparum in Mizoram.

Studies conducted in Mizoram during 1981 to 1990 have shown areas with increasing RIII level of chloroquine resistance. These foci need urgent liquidation. Sulfalene-pyrimethamine drug combination was found suitable for treatment of P. falciparum cases in these areas with only 5.3 per cent cases showing RI level of Plasmodium response. Quinine combined with sulfalene-pyrimethamine showed 100 per cent success. Amodiaquine however was similar in response to chloroquine though the mean parasite clearance time with amodiaquine was slightly better.


Management of admitted malaria cases in four major hospitals of Delhi: a case study.

A hospital-based retrospective case study of admitted patients was undertaken in four major hospitals of Delhi during 1991, with a view to assessing (i) recording and reporting system of malaria cases, (ii) diagnostic criteria being followed, (iii) management of complicated and severe malaria cases, and (iv) availability of life-saving antimalarials. The study showed that none of the hospitals either followed the international coding system for recording or adopted the National Malaria Eradication Programme guidelines for diagnostic criteria malaria, i.e. by blood smear examination. Diagnosis of malaria in three out of four hospitals was not preceded by blood examination in all cases. Only 55% of the 283 clinically suspected malaria cases were screened for malaria parasite with overall positivity of 20.14 per cent and of 38.25 per cent in examined cases. Age and sex break-up indicated that males suffered more and 65 per cent of the patients belonged to 16-40 years' age groups as compared to 38.4 per cent population falling in this age group according to 1981 census. Out of 263 recovered study cases, 13 per cent came from adjoining states while this percentage went up to 35 per cent (7 out of 20 cases) in the case of malaria deaths. Over 80 per cent of the clinically suspected cases presented with signs and symptoms of fever or fever with rigour, chills or vomiting. In 38 per cent of the cases there was a definite time lag in reporting of the cases to hospitals but most of the cases (91 per cent) were administered antimalarials within 24 h of admission.(ABSTRACT TRUNCATED AT 250 WORDS)


[The role of the HLA system in the genetics of Type I diabetes mellitus].

Major determinants of susceptibility to Type 1 (insulin-dependent) diabetes (IDDM) have been mapped to the HLA complex, near to or identical with genes encoding class II molecules. The association of IDDM with HLA-DR3 and/or DR4 antigens and the highest risk for DR3/4 heterozygotes suggest a synergistic effect of the two haplotypes. The characterization at the molecular level of the class II region has provided evidence that DQ rather than DR determinants may primarily influence the disease. In caucasians the susceptibility strongly correlates with the absence of aspartic acid at position 57 on the DQ beta chain and/or the presence of arginine at position 52 on the DQ alpha chain. The formation of a putative DQ susceptibility molecule (DQ alpha Arg52+, DQ beta Asp57-) accounts best for the disease associations when trans-complementation between alpha and beta chains encoded by different haplotypes is postulated to explain the excess of heterozygotes. Observations in other populations and in animal models indicate, however, that other residues on DQ alpha and beta chains, other class II (DR beta) molecules and non-HLA linked genes also contribute to the susceptibility. The mechanism(s) by which susceptibility determinants influence IDDM is not known. It is probably in relation with the role of class II molecules in the antigen presentation to T lymphocytes.


Increased lipid peroxidation in type 2 poorly controlled diabetic patients.

An increased lipid peroxidation, due to the altered intracellular ratio between free radicals and antioxidant systems, has been recently related to diabetes. To study the possible relationship between lipid peroxidation and metabolic control, we measured the plasma concentrations of malondialdehyde (MDA), end product of the oxidation of polyunsaturated fatty acids, in poorly and well controlled Type 2 diabetic patients. A significant increase in plasma malondialdehyde concentrations was found in poorly controlled diabetics when compared to well controlled patients (p < 0.001) and to healthy normoglycaemic subjects (p < 0.001), whereas no significant difference was observed between the two latter groups. Plasma MDA/Cholesterol and MDA/triglyceride ratios were both higher in poorly controlled diabetics than in well controlled (p < 0.005) and in normal subjects (p < 0.01 and p < 0.02 for MDA/CHOL and MDA/TG respectively). In diabetic patients a positive correlation was found between plasma MDA levels and mean daily blood glucose (p < 0.01), plasma fructosamines (p < 0.001), HbA1 (p < 0.05) and plasma triglycerides (p < 0.05), while no significant correlation was shown between plasma malondialdehyde and total cholesterol. Malondialdehyde levels were followed-up for 7 days running (T1-T7) in five poorly controlled diabetics, treated with conventional insulin therapy. This group showed normalized plasma lipid peroxide values (0.486 +/- 0.13 mumol/l, T5, M +/- SEM) 72 h after the restoration of glycaemic control (145 +/- 25 mg/dl, T2, M +/- SEM). These results confirm the increase of lipid peroxidation during Type 2 diabetes. The correlation with the degree of metabolic imbalance suggests a possible role for lipid peroxidation in the occurrence of glucose-induced macromolecular changes.


Intermediate and long-acting insulin preparations without protamine sulphate are complement activators in vitro.

Recently, we have documented an abnormal in vivo complement metabolism in Type 1 diabetic children treated with monocomponent porcine insulin Monotard MC and its correction after switch-over to human insulin Protaphane HM. This prompted us to investigate the ability of different kinds of insulin preparations to induce complement activation in vitro. Freshly collected serum samples from healthy blood donors were incubated with commercial rapid and intermediate or long-acting (by protamine sulphate (PS) or zinc) insulin preparations for 2 hours at 37 degrees C. The C3d content of the supernatants was measured by turbidimetry as a marker of C3 complement fraction consumption. Only long-acting preparations of insulins without protamine sulphate were associated with highly significant increased levels of C3d, whatever the source of insulin, animal or human. Moreover, addition of exogenous protamine sulphate was able to inhibit the C3 conversion. This effect was dose-dependent and peaked at the concentration of commercial NPH insulin preparations. The mechanism by which protamine sulphate inhibits complement activation in vitro could be related to its ability to interfere with the physical nature of the solid surfaces presented by the insulin crystals. Indeed, insulin crystals were rapidly cleared (< 5 min) in the incubated serum when small doses of protamine sulphate were added. The complement activating capacity of long-acting insulin without protamine was dose dependent, equivalent to the known complement activator Zymosan, and abolished in the presence of EDTA. In conclusion, the present study has documented the ability of some protracted insulin preparations to activate the complement system in vitro if they are devoided of protamine sulphate. On the other hand, short-acting and NPH insulins are not complement activators.


Differential effects of insulin-induced hypoglycaemia on the plasma branched-chain and non-branched-chain amino acid concentrations in humans.

In order to determine plasma amino acid concentrations during a prolonged but moderate insulin-induced hypoglycaemia, six healthy volunteers received a constant subcutaneous insulin infusion (15 mU.m-2.min-1) over a 12 hour period. The plasma glucose concentrations decreased from 4.72 +/- 0.11 to 2.83 +/- 0.07 mM at 600 minutes and then remained stable over the last 120 minutes. Plasma counterregulatory hormones (glucagon, epinephrine, growth hormone and cortisol) increased significantly between 120 and 180 minutes. The plasma concentration of all the amino acids paralleled the decrease in plasma glucose. The branched chain amino acids decreased to a greater extent in the first part of the study (0-360 min) in comparison to the essential non-branched chain aminoacids (p < 0.01), then increased significantly with a peak at 600 minutes (p < 0.05 vs 360 min) despite stable hyperinsulinaemia. These results suggests that during prolonged but moderate hypoglycaemia the counterregulatory hormones are able to antagonize partially the effects of insulin on protein metabolism, analogous to their well-known anti-insulin effects on glucose and fatty acid metabolism.


Influence of obesity and hypertriglyceridaemia on the low HDL2-cholesterol level and on its relationship with prevalence of atherosclerosis in type 2 diabetes.

High density lipoprotein subfraction 2 (HDL1)-cholesterol level is usually decreased in Type 2 (non-insulin-dependent) diabetes. A study was carried out in 251 Type 2 diabetic patients (106 males [M], 145 females [F]) and in 120 non diabetic controls in order to determine the influence of hypertriglyceridaemia and obesity on the HDL2-cholesterol level and to analyse the relationship between HDL2-cholesterol level and atherosclerosis (coronary heart disease, peripheral atherosclerosis or cerebral vascular disease), in Type 2 diabetes. Influence of hypertriglyceridaemia and obesity on HDL2-cholesterol level was studied by comparing the mean values of HDL2-cholesterol between diabetics and controls, after controlling for hypertriglyceridaemia and obesity, and by a multiple linear regression test. A stepwise logistic regression was performed to analyse the association between the prevalence of atherosclerosis and several variables: age, duration of diabetes, hypertension, cigarette smoking, body mass index, mean glycaemia, total cholesterol, triglyceride, HDL-cholesterol, HDL2-cholesterol and HDL3-cholesterol levels. In both men and women, when both of the factors (hypertriglyceridaemia and obesity) were present of when only one was, HDL2-cholesterol level was significantly lower in the diabetic population, compared with controls. But when obesity and hypertriglyceridaemia were absent, HDL2-cholesterol level, in the diabetic population, was not significantly different from controls (M: 17.9 +/- 13.3 vs 20.5 +/- 13.8 mg/dl: NS; F: 30.1 +/- 21.5 vs 27.6 +/- 14.2 mg/dl: NS).(ABSTRACT TRUNCATED AT 250 WORDS)


Short-term metabolic effects of the ACE-inhibitor benazepril in type 2 diabetes mellitus associated with arterial hypertension.

To assess the short-term metabolic effects a long-acting non-sulphydryl ACE-inhibitor benazepril on glycaemic control in Type 2 diabetes mellitus and arterial hypertension, 10 hypertensive diabetic patients treated with glibenclamide were studied in a double-blind, crossover fashion over two 10-day periods in which either benazepril (10 mg/day) or placebo was given. At the end of the 10 day treatment, both blood pressure and plasma glucose concentrations were lower after benazepril versus placebo (benazepril, blood pressure: 143 +/- 11/83 +/- 5 mmHg, plasma glucose: 7.1 +/- 1.2 mmol/l; placebo: blood pressure: 157 +/- 10/99 +/- 2 mmHg, plasma glucose: 8.2 +/- 1 mmol/l, p < 0.05). In response to an oral glucose tolerance test combined with 1 mg intravenous glibenclamide, plasma glucose levels were lower after benazepril versus placebo (0-460 min: 8.4 +/- 0.8 versus 10.5 +/- 0.9 mmol/l, p < 0.05), whereas plasma insulin, C-peptide and glibenclamide concentrations were not different. It is concluded that a short-term administration of benazepril in Type 2 diabetes mellitus reduces blood pressure and improves blood glucose control, most likely by decreasing insulin resistance.